Glycan-based drugs, therapies and biomarkers

ABSTRACT

The present disclosure discloses simple and efficient glycan- or carbohydrate-based processes or methods for the rapid identification of biological markers and therapeutic targets especially glycan-related targets of infectious diseases, cancers, autoimmune diseases, allergies, inflammation, toxicity, obesity and/or other disorders of humans, animals, plants and other organisms. Therefore, novel methods and products for the diagnosis, prevention, and treatment of such diseases obtainable based on these therapeutic targets can be developed.

This application is a continuation-in-part of PCT App. No. US09/39810,entitled “Glycan Based Array and Uses Thereof” filed on Apr. 7, 2009,which claims priority to U.S. Prov. App. 61/043,396, filed Apr. 8, 2008,entitled “Glycan Based Molecular Mimicry Array and the Uses Thereof.”This application also claims priority to U.S. Prov. App. 61/278,685,filed Oct. 9, 2009, entitled “Glycan-based drugs, therapies andbiomarkers” and to the U.S. Prov. App. No. 61/335,415, filed Jan. 7,2010, entitled “Anti-virus drugs, therapies vaccines and experimentalmodels.” The entire specification and disclosure of these fourapplications are incorporated by reference herein.

FIELD OF INVENTION

The present disclosure relates generally to the fields of biology,medicine and epidemiology, and in particular, to one or more processesfor diagnosing, preventing and/or treating infectious diseases, cancers,autoimmune diseases, allergies, toxicity, obesity and/or other disordersof humans, animals, plants and other organisms. More specifically, thepresent disclosure relates to processes for the identification oftherapeutic targets of the disorders mentioned above and the usesthereof.

BACKGROUND

Carbohydrates are an essential component of life as a structural andenergy storage component, and as stabilization, recognition, signalingand communication agents. Increasing interest in glycobiology has beenprecipitated by recent findings that cell surface carbohydrates arecritically involved in cell adhesion and, thus, in cell-cellinteraction. The advent of molecular biology in this field has enabledscientists to manipulate carbohydrate expression and study glycoproteinfunction.

Difficulties in the Study of Sugar Structures

Part of the variability seen in saccharide structures is due to the factthat monosaccharide units may be coupled to each other in many differentways, as opposed to the amino acids of proteins or the nucleotides inDNA, which are always coupled together in a standard fashion. The studyof saccharide structures is also complicated by the lack of a directtemplate for their biosynthesis, contrary to the case with proteinswhere their amino acid sequence is determined by their correspondinggene.

Saccharides are also secondary gene products and as such are generatedby the coordinated action of many enzymes in the subcellularcompartments of a cell. Thus, the structure of a saccharide may dependon the expression, activity and accessibility of the differentbiosynthetic enzymes. This means it is not possible to use recombinantDNA technology in order to produce large quantities of saccharides forstructural and functional studies as has been used extensively forprotein studies.

SUMMARY OF THE INVENTION

Aspects of the present disclosure are based on the concepts that cellsurface glycans or carbohydrates are critically involved in cell-cellinteraction; that in at least some form, glycans or carbohydrates areshared by some or all organisms during life origination and evolution;and that carbohydrates changes at different physical status. Thereof,the present disclosure illustrates simple and efficient glycan- orcarbohydrate-based processes or methods for the rapid identification ofbiological markers and therapeutic targets especially glycanrelatedtargets that are related to infectious diseases, cancers, autoimmunediseases, allergies, inflammation, toxicity, obesity and/or otherdisorders of humans, animals, plants and other organisms. The processaccording to the present disclosure in one embodiment is characterizedby the following operations:

1) the attachment and/or fixation of healthy and disease cells and/ortissues of organisms, pathogens, glycans, lectins, glycan recognitionsystems, antibodies and/or sera, herbs, small molecules and toxins (allcalled target candidates hereafter) to at least one solid carrier for anarray or microarray (called array carrier hereafter);

2) the binding of an antibody or a serum, a pathogen, a glycan, alectin, a glycan recognition system, a herb, a small molecule or a toxin(all called “detection candidates” hereafter) to the arraycarrier/carriers;

3) the detection of the binding of a detection candidate to the targetcandidates on the array carrier/carriers;

4) the detection of the biological function, pathogenesis, pharmacology,toxicity of a detection candidate with positive binding to at least oneof the targets attached on the array carrier/carriers, in animalexperiments and/or cell or tissue culture systems;

5) the application of the detection candidates relevant to infectiousdiseases, autoimmune diseases, allergies, cancers, obesity and otherdisorders determined in step 4 for the diagnosis, prevention andtreatment of these disorders; for drug discovery and delivery; forvaccine development and to the fields of epidemiology and biologyespecially developmental and evolutionary biology;

6) the identification of the therapeutic targets or markers of thehealthy and/or disease tissues and cells of organisms (attached on thearray carrier/carriers) bound by at least one detection candidate;

7) the application of the therapeutic targets or markers identified, instep 6, their derivatives (including but not limited to analogs,agonists, antagonists, variants, mutants, fragments, synthetic peptides,recombinant antigens) and any other forms of the therapeutic targets forthe diagnosis, prevention, treatment and drug delivery of infectiousdiseases, autoimmune diseases, allergies, cancers, obesity and otherdisorders related to at least one of the therapeutic targets with knownor unknown etiology and/or pathogenesis.

Accordingly, in one embodiment the present disclosure is to providesimple and efficient processes or methods for the rapid identificationof therapeutic targets; for the discovery of drugs and drug deliverysystems; for the pathogenesis studies and cause screening of infectiousdiseases, autoimmune diseases, allergies, toxicity, cancers,inflammation, obesity and other disorders of humans, animals, plants andother organisms; for development of high quality and new vaccines; foreffective control of pandemic diseases; for functional, toxic,pharmacological and pharmaceutical studies of lectins, herbs, toxins andsmall molecules; for development of animal models of autoimmunediseases, allergies, toxicity, cancers, obesity and other disorders; andfor studies of epidemiology and biology especially evolutionary biology.Numerous other objects, features and advantages of the presentdisclosure will become readily apparent from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of an example array chip.

FIG. 2 is a graphical representation of binding of plant lectins of WGAand soybean agglutinin (SBA) to tissue sections of adult mice.

FIG. 3 is a graphical representation of binding of plant lectins ofwheat germ agglutinin (WGA) and Ulex Europaeus agglutinin I (UEA I) totissue section of small intestine of healthy newborn pups and adultmice.

FIG. 4 is a graphical representation of binding of plant lectin WGA totissue sections of rhesus rotavirus (RRV) infected and uninfected mousepups.

FIG. 5 is a graphical representation of binding of an anti-RRVpolyclonal antibody and lectin WGA to tissue sections of RRV infectedand uninfected mouse pups.

FIG. 6 is a graphical representation of RRV infected mouse pups treatedwith a formulation and saline.

FIG. 7 is a graphical representation of detection of goat IgG in sera ofpups delivered to those dams injected with goat anti-RV (B) by adot-blot hybridization. Various concentrations of goat IgG were used ascontrols (A). Binding of goat IgG to goblet cells of small intestine ofthe pups were detected at age of week 1 (C) by PE-anti-goat IgG,compared to a control without antibody treatment (D).

FIG. 8 is a graphical representation of in vivo binding ofanti-respiratory syncytial virus (RSV) antibody to tissue sections ofuninfected mouse pups.

FIG. 9 is a graphical representation of histology changes induced byinjection of goat anti-RV antibodies to pregnant mice. A-B: the bileduct and gall bladder of mouse pups at week 2 (A) and week 1 (B); C:mouse pup livers at week 1; D-E: the pup bile duct at P4 and P5.

FIG. 10 is a graphical representation of survival curve of differentmouse modules of rotavirus infection.

FIG. 11 is a graphical representation of detection of binding ofanti-rotavirus antibodies (NCDV) and a proliferation marker (PCNA) totissue sections of RRV infected and uninfected mouse pups.

FIG. 12 is a graphical representation of 2009 H1N1 influenzavirus-infected newborn chicks. Chicken embryo were pre-treated at E16with saline (A), anti-2009H1N1 (California) virus serum (B),anti-seasonal H1N1 (Shanghai, 1999) virus serum (C), and a formulaconsisted of the anti-2009H1N1 (California) serum and Neu5Ac (D),followed by viral inoculation at E17.

FIG. 13 is a graphical representation of newborn chicks treated at E16with saline (A), anti-2009H1N1 (California) virus serum (B),anti-seasonal H1N1 (Shanghai, 1999) virus serum (C), anti-H3N2 (Jiangxi,2004) virus serum (D), anti-H5N1 (Anhui, 2005) virus serum (E), and ahealthy human serum pool (F).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

While the present disclosure is susceptible of embodiment in manydifferent forms, there will be described herein in detail, preferred andalternate embodiments of the present disclosure. It should be understoodhowever, that the present disclosure is to be considered anexemplification of the principles of the invention and is not intendedto limit the spirit and scope of the invention and/or claims of theembodiments illustrated.

Array Carriers and Attachment or/and Fixation of Materials and Chemicals

In the present disclosure, a solid carrier (called array carrierhereafter) for the use of array or microarray refers to an object whichcan be used for attachment of materials of organisms and chemicalsinclude but not limited to a slide, a plate, a membrane, a strip, achip, or a particle, etc., without limitation. Materials of organismsand chemicals can be attached or fixed to at least one array carrier.The methods for attachment and fixation of materials of organisms andchemicals to a solid carrier can be physical, chemical, biological andall the other ways known in the arts.

Materials and Chemicals for Primary Screening

According to the disclosure, materials of organisms and chemicalsinclude but not limited to follows.

Organism and Pathogens

In the present disclosure, the term organism refers to an individualliving system including but not limited to animals, plants, insects,fungi or micro-organisms. Based on cell type, organisms can be dividedinto the prokaryotic and eukaryotic groups. The prokaryotes aregenerally considered to represent two separate domains, called theBacteria and Archaea. Eukaryotic organisms include but not limited tohumans, animals, plants, fungi, slime mould, algae, organelles,mitochondria and (in plants) plastids, viral eukaryogenesis, etc. Morerecently a Glade, Neomura, has been proposed, by Thomas Cavalier-Smith,which groups together the Archaea and Eukarya. Cavalier-Smith alsoproposed that the Neomura evolved from Bacteria, more precisely fromActinobacteria.

A microorganism (also can be spelled as micro organism) or microbe is anorganism that is microscopic (too small to be seen by the naked humaneye). One aspect of the present disclosure relates to microorganismsincluding but not limited to beneficial microorganisms, archaea,pathogenic microorganisms responsible for illness and/or organismsrelated to life evolution. More specifically, microorganisms include butnot limited to bacteria, viruses, fungi, viroids, prions, etc.

As used herein, a “pathogen” refers to a pathogenic organism includingbut not limited to a microorganism, a parasite, an insect, a plant, andetc., withpout limitation. The term “infectious diseases” refers to thedetrimental colonization of a host organism by a foreign species.Pathogens specific to infectious diseases suitable for use in thisprocess include, but are not limited to, viruses, bacteria, parasites,fungi, viroids, prions, protozoa, and insects.

Types of pathogens include but not limited to any types of pathogens,live or dead or inactivated, fresh or dried, fixed or frozen, whole orpart or fragment, sections, smears, homogenates, lysates, and extractsof pathogens, and etc., without limitation.

Antibodies

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. Such antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, single chain,F.sub.ab, F.sub.ab′ and F(ab′).sub.2 fragments, and an Fab expressionlibrary. In general, an antibody molecule obtained from humans relatesto any of the classes IgG, IgM, IgA, IgE and IgD, which differ from oneanother by the nature of the heavy chain present in the molecule.Certain classes have subclasses as well, such as IgG.sub.1, IgG.sub.2,and others. Furthermore, in humans, the light chain may be a kappa chainor a lambda chain. Reference herein to antibodies includes a referenceto all such classes, subclasses, antibody fragments and types of humanantibody species. Natural occurring antibodies are found in blood orother bodily fluids of vertebrates.

Antibodies suitable for use in this disclosure can be specific for anyorganisms, any pathogens, any infectious agents, any glycans, anyglycoconjugates, any “self” antigens or any biological markers of anorganism, and etc., without limitation. Sera from patients diagnosedinfectious diseases, autoimmune diseases, allergies, toxicity, cancers,obesity and other disorders are also included.

Preferably antibodies to viruses suitable for use in this processinclude but not limited to any types of antibodies or antibody fragmentsto dsDNA viruses including but not limited to adenoviridea,herpesviridea, papovaviridea, poxviridea; the ssDNA viruses includingbut not limited to circoviridea, geminiviridae, parvovirinae; dsRNAviruses including but not limited to birnaviridae, reoviridea, (+)senseRNA viruses including but not limited to astroviridea, caliciviridea,coronaviridea, flaviviridea, picornaviridea, potyviridea, tabamoviridea,togaviridea; (−)sense RNA viruses including but not limited tofiloviridea, paramyxoviridea, pneumovirinae, rhabdoviridea, arenavirus,bunyaviridea, orthomyxoviridea; RNA reverse transcribing virusesincluding but not limited to retroviridea; DNA reverse transcribingviruses including but not limited to badnavirus, caulimoviridea,hepadnaviridea; satellites including but not limited to tobacco necrosisvirus satellite; hepatitis delta virus; viroids including but notlimited to potato spindle tuber viroid, and agents of spongiformencephalopathies. More specifically, antibodies to viruses include butnot limited to any types of antibodies to reovirus, rotavirus,cytomegalovirus, influenza virus including avian influenza A virus,Epstein-Barr virus, hepatitis virus, HIV, HTLV, papilloma virus, poliovirus, parainfluenza virus, measles virus, mumps virus, respiratorysyncytial virus, shipping fever virus, Western and Easternencephalomyelitis virus, Japanese B encephalomyelitis virus, Russianspring-summer encephalomyelitis virus, hog cholera virus, pox virus,rabies, virus, distemper virus, foot and mouth disease virus,rhinovirus, Newcastle disease virus, vaccinia virus; and pseudorabiesvirus, etc without limitation.

Preferably antibodies to bacteria suitable for use in this processinclude but not limited to any types of antibodies or antibody fragmentsto Gram-positive and Gram-negative bacteria, or bacilli (rod-shaped),cocci (spherical) and spirilla (curved walls), and other bacteria.Specific bacteria include but not limited to cholera, syphilis, anthrax,leprosy and bubonic plague, rickettsias, neisseria gonorrhoeae,bordetella pertussis, escherichia coli, salmonella enterica, vibriocholerae, pseudomonas aeruginosa, yersinia pestis, francisellatularensis, haemophilus influenzae, purple sulfur bacteria, helicobacterpylori, campylobacter jejuni, bacillus anthracis/cereus/thuringiensis,clostridium tetani, clostridium botulinum, staphylococci, streptococci,pneumococci, streptococcus pneumoniae, mycoplasmas, bacteroidesfragilis, mycobacterium tuberculosis, mycobacterium leprae,corynebacterium diphtheriae, treponema pallidum, borrelia burgdorferi,chlamydia trachomatis, chlamydia psittaci, phycocyanin, phycoerythrin,mitochondria, chloroplasts, etc., without limitation.

Cells, Tissues and Organs of Organisms

As used herein, the term of disease refers to an abnormal condition ofan organism that impairs bodily functions, associated with specificsymptoms and signs in human beings or animals, “disease” is often usedmore broadly to refer to any condition that causes discomfort,dysfunction, distress, social problems, and/or death to the personafflicted, or similar problems for those in contact with the person orthe animal. In this broader sense, it sometimes includes disabilities,disorders, syndromes, infectious diseases, isolated symptoms, deviantbehaviors, and atypical variations of structure and function, while inother contexts and for other purposes these may be considereddistinguishable categories. Types of diseases include but not limited toinfectious diseases, cancers, autoimmune diseases, allergies, toxicity,obesity and/or other disorders of humans, animals, plants and otherorganisms.

Types of healthy or normal and disease cells, tissues and/or organs ofeukaryotes according to the present disclosure can be any types of cellsbeing cultured in vitro including but not limited to various cell linesand primary cells known in the art; any types of cells being obtainedfrom fresh tissues; any types of fragments, sections or smears of fresh,frozen or fixed tissues or organs; extracts or homogenates or lysates ofcells or tissues or organs, any types of organ parts, or any other typesof cells, tissues or organs, without limitation.

Healthy and disease cells or tissues or organs of humans, animals orplants include their either part or intact period of life time fromembryo, fetal, newborn, young child to adult. The healthy and nothealthy tissues or organs of humans and animals can be but not limitedto epithelium and glands; connective tissue; muscle including smooth,skeletal and cardiac muscle; nervous tissue including central nervoussystem (CNS) and peripheral nervous system (PNS); cartilage, bone andjoints; extracellular matrix; blood and hemopoiesis; bone marrow;cardiovascular system including heart, arteries, capillaries and veins;respiratory system including lungs, bronchial tree, alveolar duct andalveoli, digestive system including oral cavity, esophagus, stomach,small intestine (duodenum, jejunum, and ileum), and large intestine(cecum, colon, rectum, anal canal and appendix), salivary glands,pancreas, liver, bile duct and gallbladder; urinary system includingkidneys, ureter, bladder, and urethra; female reproductive systemincluding ovaries, oviducts, uterus and vagina; male reproductive systemincluding testes, genital ducts, penis, seminal vesicles, prostategland, and bulbourethral glands; lymphois (immune) system includinglymph nodes, thymus and spleen; endocrine glands including pineal body,pituitary gland, thyroid gland, parathyroid glands and suprarenalglands; integument including skin and its appendages, sweat glands,sebaceous glands, hair and nails.

Glycans and Glycoconjugates

The term glycan refers to a polysaccharide, or oligosaccharide. Anoligosaccharide is a saccharide polymer containing a small number(typically three to ten) of component sugars, also known as simplesugars. Glycans usually consist of O- or N-glycosidic linkages ofmonosaccharides to compatible amino acid side chains in proteins or tolipid moieties. Two types of glycosylation exist: N-linked glycosylationto the amide nitrogen of asparagine side chains and O-linkedglycosylation to the hydroxy oxygen of serine and threonine side chains.Other glycans include but not limited to O-GlcNAc, GAG Chain,glycosaminoglycans, and glycosphinglipid. Monosaccharides include butnot limited to fructose, glucose, mannose, fucose, xylose, galactose,lactose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids.O- and N-linked glycans are very common in eukaryotes but may also befound, although less commonly, in prokaryotes. Glycans can be foundattached to proteins as in glycoproteins and proteoglycans. They aregenerally found on the exterior surface of cells. Sialic acid is ageneric term for the N- or O-substituted derivatives of neuraminic acid,a nine-carbon monosaccharide. Some members of this group includeN-acetylneuraminic acid (Neu5Ac or NANA), 2-Keto-3-deoxynononic acid(Kdn), N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc),N-Acetylmannosamine (ManNAc), and N-Glycolylneuraminic acid (Neu5Gc).Sialic acids are found widely distributed in animal tissues and inbacteria, especially in glycoproteins and gangliosides. The amino groupbears either an acetyl or a glycolyl group. The hydroxyl substituentsmay vary considerably: acetyl, lactyl, methyl, sulfate and phosphategroups have been found. Sialic acid rich glycoproteins bind selectin(Ctype lectin) in humans and other organisms.

Glycanconjugates

In the present disclosure, the term glycan also refers to thecarbohydrate portion of a glycoconjugate, include but not limited toglycoproteins, glycolipids, proteoglycans and glycophosphosphingolipidsor any other known or unknown glycoconjugates. Glycoconjugates are foundpredominantly on the outer cell wall and in secreted fluids.Glycoconjugates have been shown to be important in cell-cellinteractions due to the presence on the cell surface of various glycanbinding receptors in addition to the glycoconjugates themselves.http://en.wikipedia.org/wiki/Glycobiology-_note-Ma_(—)2004

The term of proteoglycans represent a special class of glycoproteinsthat are heavily glycosylated. They consist of a core protein with oneor more covalently attached glycosaminoglycan (GAG) chain(s). Theseglycosaminoglycan chains are long, linear carbohydrate polymers that arenegatively charged under physiological conditions, due to the occurrenceof sulfate and uronic acid groups. Proteoglycans can be categoriseddepending upon the nature of their glycosaminoglycan chains. Thesechains include but not limited to chondroitin sulfate and dermatansulfate; heparin and heparan sulfate; keratan sulfate. Proteoglycans canalso be categorised by size. Examples of large proteoglycans areaggrecan, the major proteoglycan in cartilage, and versican, present inmany adult tissues including but not limited to blood vessels and skin.The small leucine rich repeat proteoglycans (SLRPs) include but notlimited to decorin, biglycan, fibromodulin and lumican.

The term of glycolipids refers to carbohydrate-attached lipids. Theyoccur where a carbohydrate chain is associated with phospholipids on theexoplasmic surface of the cell membrane. They extend from thephospholipid bilayer into the aqueous environment outside the cell whereit acts as a recognition site for specific chemicals as well as helpingto maintain the stability of the membrane and attaching cells to oneanother to form tissues. Glycolipid includes but not limited togalactolipids, sulfolipids (SQDG), glycosphingolipids, cerebrosides,galactocerebrosides, glucocerebrosides, glucobicaranateoets,gangliosides, globosides, sulfatide, glycophosphosphingolipids, or anyother known or unknown glycolipids.

Glycan Recognition System

Glycans, which are assumed to have been first synthesized in the form ofsimple homopolysaccharides (amylose, cellulose, etc.), are understood tohave evolved into more complex hetero-polysaccharides. This evolution isassumed to have triggered the advent of proteins (lectins) related tothe “recognition system of glycans” that recognizes each structure,identifies molecules, introduces biological signaling and facilitatesinfectious diseases. The synthesis system and the recognition system ofglycans depend on each other and are still considered to be undergoingco-evolution.

Recognition system of glycans includes but not limited to lectinsincluding animal-, plant-, and pathogen-lectins, enzyme containingcarbohydrate recognition domain (CRD), antibodies against glycans,cytokines, chaperone and transport proteins, microbialcarbohydrate-binding croteins, clycosaminoglycan-binding proteins, orany other known or unknown glycan recognition systems, withoutlimitation.

Lectins are sugar-binding proteins which are highly specific for theirsugar moieties. In the present disclosure, lectins include but notlimited to animal lectins, plant lectins, pathogen lectins, and anyother know or unknown lectins. Lectins occur ubiquitously in naturewhich typically contains an evolutionarily conservedcarbohydrate-recognition domain. Lectins are known to play importantroles in the immune system by recognizing carbohydrates that are foundexclusively on pathogens, or that are inaccessible on host cells.Pathogenic lectins from virus, bacteria, protozoa and insects areinvolved in infection through their sialic acid-recognizing activity.

Animal lectins include but not limited to C-type, M-type, L-type,P-type, R-type, I-type, F-type, F-box H-type lectins, galectins,pentraxin, spider toxin, tachylectin, chitin-binding protein,chitinase-like lectins, TIM-lectin, calnexin-calreticulin, ficolins,fucolectin, intelectins, and any other types of know or unknown animallectins.

Plant lectins include but not limited to β-prism I lectin, β-prism IIlectin, β-trefoil lectin, knottin, legume lectin, fructose-, mannose-,glucose-, fucose-, galactose-, Nacetylgalactosamine-, andN-acetylglucosamine-specific lectins, and any other types of know orunknown plant lectins.

Pathogen lectins include but not limited to bacterial lectins, viruslectins and fungal lectins. Bacterial lectins include but not limited toAB 5 toxin, bacterial neurotoxin, staphylococcal toxin, pili adhensin,cyanobacterial lectins, 1-Ca β-sandwich, 2-Ca βsandwich, β-propeller,toxin repetitive domain. Virus lectins include but not limited to coatprotein, hemmagglutinin, tailspike protein, capsid spike protein andfiber knob. Fungal lectins include but not limited to Ig-like,actinoporin-like, β-trefoil pore forming lectins, galectin, 6bladedβ-propeller, and 7-bladed β-propeller, and any other types of know, orunknown pathogen lectins.

Animal glycan-recognizing proteins including but not limited to twogroups—lectins and sulfated glycosaminoglycan (SGAG)-binding proteins.The biosynthesis of structurally complex GAG is regulated and itsdiverse sulfation pattern is formed organ- and tissue-specifically aswell as temporally during growth and development. Proteins other thanantibodies and T-cell receptors that mediate glycan recognition viaimmunoglobulin (Ig)-like domains are called “I-type lectins.” The majorhomologous subfamily of I-type lectins with sialic acid (Sia)-bindingproperties and characteristic amino-terminal structural features arecalled the “Siglecs” (Sia-recognizing Ig-superfamily lectins).

Mucins can be sialic acid-containing glycoproteins. Mucins are secretedin the mucus of the respiratory and digestive tracts. Mucin genes encodemucin monomers that are synthesized as rod-shape apomucin cores that arepost-translationally modified by exceptionally abundant glycosylation.Two distinctly different regions are found in mature mucins: 1) Theamino- and carboxy-terminal regions are very lightly glycosylated, butrich in cysteines, which are likely involved in establishing disulfidelinkages within and among mucin monomers. 2) A large central regionformed of multiple tandem repeats of 10 to 80 residue sequences in whichup to half of the amino acids are serine or threonine. This area becomessaturated with hundreds of O-linked oligosaccharides. N-linkedoligosaccharides are also found on mucins, but much less abundantly. Atleast 19 human mucin genes have been distinguished by cDNA cloning—MUC1,2, 3A, 3B, 4, SAC, 5B, 6-9, 11-13, and 15-19. The major secreted airwaymucins are MUC5AC and MUC5B, while MUC2 is secreted mostly in theintestine but also in the airway. Increased mucin production occurs inmany adenocarcinomas, including cancer of the pancreas, lung, breast,ovary, colon, etc. Mucins are also over expressed in lung diseases suchas asthma, bronchitis, COPD or cystic fibrosis.

Herbs and Traditional Chinese Herbs

As used herein, an herb refers to a plant that is valued for qualitiessuch as medicinal properties, flavor, scent, or the like. In the presentdisclosure, traditional Chinese herbs include but not limited to allherbs listed in Bencao Gangmu (traditional Chinese:

simplified Chinese:

; pinyin: B{hacek over (e)}nc{hacek over (a)}oGāngmù; Wade-Giles:Pen-ts'ao Kang-mu), also known as Compendium of Materia Medica, which isChinese materia medica work written by Li Shizhen in Ming Dynasty. It isa work epitomizing materia medica (

) in Ming Dynasty. It lists all the plants, animals, minerals, and otherobjects that were believed to have medicinal properties.

Specifically, fundamental traditional Chinese herbs include but notlimited to: Agastache rugosa—huò xiāng (

), Alangium chinense—bā ji{hacek over (a)}ofēng (

), Anemone chinensis (syn. Pulsatilla chinensis)—bái tóu weng (

), Anisodus tanguticus—shān làng dàng (

), Ardisia japonica—z{hacek over (i)} jīn niú (

), Aster tataricus—z{hacek over (i)} w{hacek over (a)}n (

), Astragalus propinquus (syn. Astragalus membranaceus)—huáng qí (

) or b{hacek over (e)}i qí (

), Camellia sinensis—chá shù (

) or cháyè (

), Cannabis sativa—dàmá (

), Carthamus tinctorius—hóng huā (

), Cinnamomum cassia—ròu gùi (

), Cissampelos pareira—xi shēng téng (

) or (

), Coptis chinensis—du{hacek over (a)}n è huáng lián (

), Corydalis ambigua—yán hù su{hacek over (o)} (

), Croton tiglium—bā dòu (

), Daphne genkwa—yuán huā (

), Datura metel—yáng jīn huā (

), Datura stramonium (syn. Datura tatula) [13]—z{hacek over (i)} huā màntuó luó (

), Dendrobium nobile shí hú (

) or shí hú lán (

), Dichroa febrifuga [14]—cháng shān (

), Ephedra sinica—c{hacek over (a)}o má huáng (

), Eucommia ulmoides—dù zhòng (

), Euphorbia pekinensis [15]—dàj{hacek over (i)} (

), Flueggea suffruticosa (formerly Securinega suffruticosa)—yī yè qiū (

), Forsythia suspensa liánqiào (

), Gentiana loureiroi—dì dīng (

), Gleditsia sinensis—zào jiá (

), Glycyrrhiza uralensis—gānc{hacek over (a)}o (

), Hydnocarpus anthelminticus (syn. H. anthelminthica)—dà fēng z{hacekover (i)} (

), Ilex purpurea—dōngqīng (

), Leonurus japonicus—yì m{hacek over (u)} c{hacek over (a)}o (

), Ligusticum wallichii [18]—chuān xiōng (

), Lobelia chinensis—bàn biān lián (

), Phellodendron amurense—huáng b{hacek over (a)}i (

), Platycladus orientalis (formerly Thuja orientalis)—cèb{hacek over(a)}i (

), Pseudolarix amabilis—jīn qián sōng (

), Psilopeganum sinense—shān má huáng (

), Pueraria lobata—gé gēn (

), Rauwolfia serpentina—shégēnmù (

), cóng shégēnmù (

), or yìndù shé mù (

), Rehmannia glutinosa—dìhuáng (

) or gān dìhuáng (

), Rheum officinale—yào yòng dà huáng (

), Rhododendron tsinghaiense—Qīng h{hacek over (a)}i dù juān (

), Saussurea costus—yún mù xiāng (

), Schisandra chinensis—w{hacek over (u)} wèi zi (

), Scutellaria baicalensis—huáng qín (

), Stemona tuberosa—b{hacek over (a)}i bù (

), Stephania tetrandra fáng j{hacek over (i)} (

), Styphnolobium japonicum (formerly Sophora japonica)—huái (

), huái shù (

), or huái huā (

), Trichosanthes kirilowii—guā lóu (

), and Wikstroemia indica—li{hacek over (a)}ogē wáng (

), Isatis indigotica—ban lan gen (

), Yun nan bai yao (

), Eclipta prostrate herb dan shen (

), Taraxacum mongolicum herb—pu gong ying (

), Ginseng—ren shen (

), Rehmannia glutinosa/foxglove root prep.—shu di huang (

), Panta Teapills—jiao gu lan (

), Dioscorea opposite/Chinese yam rhizome—shan yao (

), Paeonia suffruticosa/peony tree root-bark—mu dan pi (

), Poria cocos fungus/mushroom filaments—fu ling (

), Alisma plantago aquatica/water plantain rhizome—Ze xie (

), Cornus officinalis/dogwood tree fruit—shan zhu yu (

), Cinnamomum cassia/cinnamon bark—rou gui (

), Aconitum carmichaeli root prep.—Shu fu zi (

), Codonopsis root—dang shen (

), Eleuthro root—ci wu jia (

), Cordyceps—dong chong xia cao (

), Reshi/Mushroom of Immortality—ling zhi (

), Polygonum multiflorum root—(

), Coix lachrymal jobi/Seeds of Jobs Ears seed—yi yi ren (

), Cinnamomum cassia/cinnamon twig—gui zhi (

), Zingiber officinal rhizome. Ginger root—sheng jiang (

), Paeonia lactiflora/white peony root—bai shao (

), Angelica sinensis root dang gui (

), Ledebouriella divaricata root—fang feng (

), Poria cocos fungus—fuling (

), Eucommia ulmoides bark—du zhong (

), Atractylodes lancea rhizome e-cang zhu (

), Platycodon grandiflorum/ballon flower root—jie geng (

), Boswellia carterii—ru xiang (

), Commiphora myrrha/myrrh resin—mo yao (

), Corydalis yanhusuo/fumewort rhizome—yan hu suo (

), Prunus persica/peach seed—tao ren (

), Deer antler—lu rong (

), Atractylodes macrocephala—bai zhu (

), Mentha halocalyx/field mint hearb—bohe (

), Bupleurum chinense root—chai hu (

), Forsythisia suspense fruit—lian qiao (

), Angelica dahurica root bai zhi (

), Citrus reticulate/citrus peel—chen pi (

), Ziziphus jujube/Chinese date fruit—da zao (

), Chrysanthemum morifolium flower—ju hua (

), Ziziphus spinosa/sour jujube seed suan zao ren (

), Dioscorea opposite/Chinese yam rhizome—shanyao (

), Buckwheat—qiao mai (

), Pinellia ternata rhizome—ban xia (

).

Inorganic Ions and Small Molecules

The inorganic ions in the present disclosure include mineral nutrientsthat include but not limited to elements boron, copper, manganese, zinc,molybdenum, sulphur, iron, calcium, potassium, nitrate, phosphate,chloride, etc., without limitation. The small molecules in the presentdisclosure include but not limited to glycan binding peptides,carbohydrate chains which are also called aliphatic hydrocarbons andhave the collective formula C_(n)H_(n+2); structural isomers which sharethe same hydrocarbon formula but have different structures; unsaturatedhydrocarbons in which carbohydrate chains containing multiple bonds;alcohols which are aliphatic carbon compounds that carry on or morehydroxyl-groups directly linked to a carbon atom; aldehydes that ofsecondary alcohols to ketones; organic acid which are produced by theoxidation of an aldehyde group; esters which are produced by acondensation reaction of an alcohol and an acid, or any other known orunknown small molecules, without limitation.

Toxins

The toxins in the present disclosure include but not limited toapitoxin, exotoxin, endotoxins, cyanotoxins, necrotoxins, hemotoxin,mycotoxin, neurotoxin, phototoxin, toxicophore, toxoid, venom, ricinis,or any other known or unknown toxins, without limitation.

A toxin is a poisonous substance produced by living cells or organismsthat is active at very low concentrations. Toxins can be smallmolecules, peptides, or proteins and are capable of causing disease oncontact or absorption with body tissues by interacting with biologicalmacromolecules such as enzymes or cellular receptors. Toxins varygreatly in their severity, ranging from usually minor and acute (as in abee sting) to almost immediately deadly (as in botulinum toxin).Biotoxins vary greatly in purpose and mechanism, and can be highlycomplex (the venom of the cone snail contains dozens of small proteins,each targeting a specific nerve channel or receptor), or relativelysmall protein.

Primary Screening

The materials and chemicals for primary screening as described above canbe used as either targets or detection reagents in the process ofprimary screen. They are called “target candidate” or “detectioncandidate” hereafter.

Target Candidates

Target candidates can be attached or fixed on to a carrier as mentionedabove in various combinations depending on needs. The combinations oftarget and detection candidates include but not limited to those listedin Table 1.

TABLE 1 Combinations of target and detection candidate binding TargetsLectins/ Small Cells/ Detection Antibodies Glycans GRS Pathogens Herbsmolecules Toxins Tissues Antibodies + + + + + + + Glycans + + + + + + +Lectins/GRS + + + + + + + Pathogens + + + + + + + Herbs + + + + + + +Small molecules + + + + + + + Toxins + + + + + + +Cells/Tissues + + + + + + + Note: Glycans include glycans and glycanconjugates. GRS = Glycan Recognition Systems

In table 1, tissues include but not limited to various healthy anddisease cells and tissues 10 of organisms and herbs include but notlimited to traditional Chinese herbs, as described above. The symbol “+”means binding of two candidates.

Type I: Target candidates include glycans, pathogens and healthytissues. Glycans include at least but not limited to fructose, glucose(Glc), mannose (Man), fucose (Fuc), xylose (Xyl), galactose (Gal),lactose, glucosamine (GlcN), galactosamine (GalN), mannosamine (ManN),N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc),N-Acetylmannosamine (ManNAc), N-Acetylneuraminic acid (Neu5Ac),N-Glycolylneuraminic acid (Neu5Gc), 2Keto-3-deoxynononic acid (Kdn),glucuronic acid (GlcA), galacturonic acid (GalA), mannuronic acid(ManA), and Iduronic acid (IdoA). Healthy tissues include but notlimited to various cells or tissues from at least one healthy organisminclude but not limited to humans, animals, plants, or other organisms.

Detection candidates for binding to the Type-I target candidates includebut are not limited to glycans, antibodies, lectins including plantlectins or other glycan recognition systems, herbs, small molecules ortoxins, depending on needs.

Type II: Target candidates include glycans (as described in Type I),pathogens and disease tissues. Disease tissues include but not limitedto various cells or tissues from at least one organism with at least oneof infectious diseases, cancers, autoimmune diseases, allergies,toxicity, obesity or other disorders. Detection candidates are as sameas described in Type I.

Type III: Target candidates include glycans, pathogens, healthy anddisease tissues (as described in Type I and Type II). Detectioncandidates are as same as described in Type I.

Type IV: Target candidates include at least one kind of targetcandidates including but not limited to antibodies, lectins includingplant lectins or other glycan recognition systems, herbs, smallmolecules and toxins, depending on needs.

Detection candidates include but are not limited to glycans, pathogens,and extracts or homogenates or lysates of healthy or disease cells ortissues or organs of an organism, depending on needs.

Type V: Target candidates include any two kinds of target candidates asdescribed in type

IV. Detection candidates are as same as described in type IV.

Type VI: Target candidates include any three kinds of target candidatesas described in type IV. Detection candidates are as same as describedin type IV.

Type VII: Target candidates include any four kinds of target asdescribed in type IV. Detection candidates are as same as described intype IV.

Type VIII: Target candidates include antibodies, lectins including plantlectins or other glycan recognition systems, herbs, small molecules andtoxins. Detection candidates are as same as described in type IV.

Other combinations of target and detection candidates depending on needsare also included.

Binding of Detection Candidates to Target Candidates

One aspect of the present disclosure relates to binding of a detectioncandidate to an array carrier with at lease one combination of targetcandidates as mentioned above. The detection candidate can be eitherpurified or conjugated with a moiety such as biotin, fluorescents or anyother detectable means known in the art. A secondary or third reagentcan be used if necessary for the detection of the candidate/carrierbinding in various ways known in the art.

The patterns of a detection candidate binding to the target candidateson an array carrier include but not limited to follows.

Type I-III Assays

Type I, Type II and Type III arrays are useful tools for the rapididentification of antigens in one organism that mimic with antigens ofat least another organism especially pathogenic organisms or infectiousagents; and determine the nature of the therapeutic targets. Examplesinclude:

-   -   a. A glycan binds to at least one pathogen: the glycan is a        potential biding site of the pathogen, and any healthy and/or        disease tissues of an organism with the glycan as part of their        cells or tissues structures can be the target of the pathogen.        The pathogen is the potential cause of the disorders relevant to        these tissues or organs.    -   b. A glycan binds to more than one pathogen: the pathogens and        the glycan are candidates for binding site-, or        receptor/ligand-, or anti-multiple pathogen-vaccines.    -   c. A detection candidate binds to at least one pathogen and at        least one healthy and/or disease tissues of an organism: there        is a therapeutic target sharing by the pathogen and the tissue.

If the detection candidate is an antibody against a pathogen, thepathogen and the antibody are potential causes of an autoimmune disease,an allergy, a toxin-relevant biological injury or another diseasetargeting the tissue or organ. The antibody is a potential candidate fordiagnosis, antibody prevention and therapy, drug delivery tool of therelevant disorders. For example, an anti-rotavirus (RV) antibody bindsto N-Acetyl-D-Glucosamine which is expressed on heart, lung and smallintestine of healthy mice (FIG. 2 and FIG. 3) and inflammatory (FIG. 4)or proliferating (FIG. 5) cells. Thus, anti-RV antibodies can be aninflammatory inducer and a cause of autoimmune diseases or cancers ofthose tissues or organs.

If the detection candidate is a glycan, a plant lectin (i.e.,mannose-binding lectin) or another glycan recognition systems, a herb, asmall molecule or a toxin, the detection candidate is a potential causeof an autoimmune disease, an allergy, a toxin-relevant biological injuryor another disease targeting the tissue or organ; or a potentialcandidate for diagnosis, prevention, therapy, drug and drug deliverytool of the relevant disorders. For example, wheat germ agglutinin (WGA)which specifically recognizes N-Acetyl-D-Glucosamine can be a cause ofthe disorders of the tissues or organs expressingN-Acetyl-D-Glucosamine.

d. A candidate binds to at least one pathogen, at least one healthyand/or disease tissue of an organism and at least one glycan: the glycanis the potential epitope of the therapeutic target sharing by thepathogen and the tissue. The glycan and its derivatives are potentialcauses of disorders relevant to the therapeutic target, or potentialcandidates for diagnosis, prevention, therapy, drug and drug deliverytool of the relevant disorders.

Type IV-VIII Arrays

Type IV, Type V, Type IV, Type-VII and Type-VIII arrays are useful toolsfor the rapid screening of causes, drugs and drug delivery tools fordiagnosis, prevention and therapy of the disorders relevant to the atherapeutic target and/or a pathogen. Examples include:

-   -   a. A target candidate of Type IV-VIII arrays is bound by a        glycan: the target candidate is a potential cause of disorders        relevant to a therapeutic target with the glycan as an epitope;        and the target candidate can be used for diagnosis, prevention,        therapy, drug and drug delivery tool of the disorders relevant        to the therapeutic target.    -   b. A target'candidate of Type IV-VIII arrays is bound by a        pathogen (i.e., a virus): the target candidate is a potential        candidate for diagnosis, prevention, therapy, drug and drug        delivery tool of the disorders relevant to the pathogen.    -   c. A target candidate of Type IV-VIII arrays is bound by an        extracts or homogenates or lysates of healthy or disease cells        or tissues or organs of an organism: the target candidate is a        potential candidate for diagnosis, prevention, therapy, drug and        drug delivery tool of the disorders relevant to the cell or        tissue or organ of the organism.    -   d. A glycan binds to at least two different antibodies against        different pathogens (i.e., antibodies against RSV and antibodies        against influenza viruses): the pathogens are candidates for        binding site- or anti-multiple pathogen-vaccines.    -   e. A pathogen (i.e., RSV) binds to more than one antibodies        against different pathogens (i.e., antibodies against RSV and        antibodies against influenza viruses): the pathogens are        candidates for binding site-, or receptor/ligand-, or multiple        pathogen-vaccines.

Other arrays with other combinations of target and detection candidatesdepending on needs are also included.

Purification and Identification of a Therapeutic Target

A biological target in one embodiment is a biopolymer such as a proteinor nucleic acid whose activity can be modified by an external stimulus.The definition is context-dependent and can refer to the biologicaltarget of a pharmacologically active drug compound, or the receptortarget of a hormone (like insulin). The implication is that a moleculeis “hit” by a signal and its behavior is thereby changed. Biologicaltargets are most commonly proteins such as enzymes, ion channels, andreceptors. The term biological target is frequently used inpharmaceutical research to describe the native protein in the body whoseactivity is modified by a drug resulting in a desirable therapeuticeffect. In this context, the biological target is often referred to as adrug target. A biomarker is a substance used as an indicator of abiologic state. It is a characteristic that is objectively measured andevaluated as an indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention. Inthe present disclosure, a therapeutic target includes but not limited toa biological target, a drug target, a biomarker, and a pathogen bindingsite, which is related to a disorder of an organism.

In another embodiment, a simple method for identification of afunctionally important therapeutic target comprises using a selectedcandidate (i.e., an antibody preferably monoclonal antibody against apathogen). This approach may eliminate laborious screening work for aninterested antigen as regularly used in the filed of proteinpurification. According to the present disclosure, sera, lysates orextract of available related cells, tissues and/or organs of humans,animals or plants as mentioned above, can be used for purification of atherapeutic target in a variety of ways well known to those of ordinaryskill in the art.

Identification of the sequence or structure of a therapeutic target, keymolecules being related to the binding of a therapeutic target and agiven candidate, derivatives of a therapeutic target in a variety ofways well known to those of ordinary skill in the art is also includedin the present disclosure.

The therapeutic targets according to the present disclosure can be aglycoprotein; glycan; polypeptides; polysaccharides; oligosaccharides;lipid, glycolipid; carbohydrate; lectin, selectin; mucin; hemagglutinin,collagen, keratin, receptor including viral receptors, toll-likereceptor; cellular component; oncogene product; fragments of mammaliancells there from including tumor cells, or any other substance withoutlimitation.

A feature of the therapeutic targets according to one embodiment of thepresent disclosure is that a therapeutic target can be either preventiveor pathogenic depending on the features of the target as describedherein.

Cellular or Tissue Culture Assays and Animal Experiments

In one embodiment, a candidate showing positive binding in the primaryscreening may be subject to following process.

Cellular or Tissue Culture Assays

According to one embodiment of the present disclosure, a cell or tissueculture assay can be used to determine the functional and pathogeniccharacteristics of a selected candidate or a combination of at least twoselected candidates. That is whether a target or detection candidate isa binding site of a pathogen, or whether a target or detection candidateinduces significant biological disorders. The cell or tissue cultureassay can be also used to determine the pharmacological, kinetics, andtoxic effect of a selected candidate or a combination of at least twocandidates determined through the primary screening of an array.Examples include:

-   -   a. Cell lines sensitive to infectious pathogens known in the        art, and also primary healthy cells or tissues or organs (i.e.,        targets of viral infectious diseases) can be cultured with a        selected candidate or a combination of at least two selected        candidates at various dosages for a period time sufficient for        the candidate to bind to its target existing on the cells or        tissues or organs, the free candidate not binding to the target        should be washed off, and the cells or tissues or organs are        treated with the pathogen (i.e., a virus strain) for a period        time sufficient for the pathogen to enter into the target cells.        The infection of the pathogen can be detected in a variety of        ways well known to those of ordinary skill in the art (i.e.,        determination of the titer of a virus strain in the culture        supernatant).

Alternatively, the same cellular or tissue culture system can becultured with an infectious pathogen at a given dosages for a periodtime sufficient for the pathogen enter into the target cells, the freepathogen remaining in the culture should be washed off, and the cells ortissues or organs are cultured with a selected candidate or acombination of at least two selected candidates at various dosages. Theinfection of the pathogen can be detected as described above.

In the case that the target is the binding site or factors related tothe entry or infection of the pathogen, the selected candidate or thecombination of at least two selected candidates can block the target andprevent the entry or infection of the pathogen into the cells or tissuesor organs. Thereof the cells or tissues or organs with treatment of theselected candidate or the combination of at least two selectedcandidates before and after infection will not be or lightly infected bythe pathogen. This method can be also used for rapid discovery of drugsand drug delivery systems relevant to infectious diseases.

-   -   b. The same cellular or tissue culture system as described above        in ‘a’ can be cultured with a selected candidate or a        combination of at least two selected candidates alone at various        dosages for a period time. The effect of a candidate a        combination of at least two candidates on cellular        proliferation, signal transduction, apotosis, necrosis and other        functions of the cells or tissues or organs can be determined in        a variety of ways well known to those of ordinary skill in the        art.

This method can be used for the functional and pathogenesis studies of acombination of at least two selected candidates, as well as causescreening of autoimmune diseases, allergies, inflammation, toxicitycancers, obesity and other disorders; for toxic, pharmacological andpharmaceutical studies of a selected candidate; for rapid discovery oftarget-based drugs and drug delivery systems relevant to thosedisorders.

-   -   c. Disease cell lines known in the art (i.e., cell lines derived        from tumor tissues), and also primary disease cells or tissues        or organs (i.e., tumor cells or tissues) can be cultured with a        selected candidate or a combination of at least two selected        candidates at various dosages for a period time. The effect of        the candidate or the combination of the at least two candidates        on cellular proliferation, signal transduction, apotosis,        necrosis and other functions of the disease cells or tissues or        organs can be determined in a variety of ways well known to        those of ordinary skill in the art.

This method can be used for the pathogenesis studies of a selectedcandidate or a combination of at least two selected candidates, as wellas cause screening of autoimmune diseases, allergies, cancers,inflammation, obesity and other disorders; for toxic, pharmacologicaland pharmaceutical studies of a selected candidate or a combination ofat least two selected candidates; for rapid discovery of target-baseddrugs and drug delivery systems relevant to those disorders.

Animal Experiments

Another subject of the present disclosure is the use of animalexperiments to determine the functional and pathogenic characteristicsof a selected target or a combination of at least two selectedcandidates. That is whether a therapeutic target is a binding site of apathogen, or whether a therapeutic target induces significant biologicaldisorders in an organism. Animals at various ages (i.e., embryo, fetus,newborn, infant and adult) can be used to evaluate characteristics oftherapeutic targets with a strong expression pattern in the embryo orfetus that decreases with growth.

The animal experiments can be also used to determine the pharmacologicaland toxic effect of a candidate or a combination of at least twocandidates determined through the primary screening for rapid discoveryof target-based drugs and drug delivery systems relevant to infectiousdiseases, autoimmune diseases, allergies, cancers, inflammation,toxicity, obesity and other disorders.

All involved animals are checked daily for symptoms of a disorder,death, etc. Blood and the target and control organs or tissues of theanimals (determined in primary screening) are collected at determinedtime point (i.e., day 1, 3, 5, 7, 14, 21, 28 after a treatment). Thecollected samples are used to evaluate the effect of a candidate or acombination of at least two candidates on inflammation, pathogenesis,toxicity, cellular proliferation, signal transduction, apotosis,necrosis and other functions of the target tissues or organs of theanimal in a variety of ways well known to those of ordinary skill in theart (i.e., histology changes). Animals being treated with candidates arealso compared to control animals without candidate treating fordetection of pathogens and/or binding of candidates to target tissuesand/or organs. Examples include:

-   -   a. A selected candidate or a combination of at least two        selected candidates can be administered to an animal (i.e., a        mouse) for a period of time sufficient for the candidate or the        combination of the at least two candidates to bind to or        interact with the target of the animal. The animal is then        challenged by the pathogen (i.e., a virus). In the case that the        target is the binding site or factors related to the entry of        the pathogen, the candidate or the combination of the at least        two candidates can block or affect the binding site and prevent        the entry of the pathogen into the target cells of the animal.        Thereof the animal will not be or lightly affected by the        pathogen. The same animal model can also be used to determine        the therapeutic effect of the selected candidate or the        combination of the at least two candidates on the relevant        disease by administering a selected candidate or a combination        of at least two candidates to an animal after the challenge of        the pathogen. Animals such as mice can be treated with the        candidate or the combination of the at least two candidates at a        low dosage within the range that yields efficacy of blocking        without much extras, with the ordinary skill in the art; virus        inoculation can be performed next day. The selected candidate or        the combination of the at least two candidates can be also        administrated one day after virus inoculation. Rest of        experiments including evaluate symptoms of infection,        determining viral titers can be performed in the ordinary skill        in the art.    -   b. Alternatively, the same animal can be treated by a selected        candidate or a combination of at least two selected candidates        alone at various dosages. A selective candidate or a combination        of at least two selected candidates can be administered        intravenously, intraperitoneally, intramuscularly,        subcutaneously, intracavity, transdermally, inhalantly or with        other approaches. A selective candidate or a combination of at        least two selected candidates can be administered into a        pregnant animal and be detected in the newborns or infants        delivered to the mother. If administration of a selected        candidate a combination of at least two selected candidates        alone induces a significant biological disorder in an animal,        the target and the candidate is identified as pathogenic, and        the animal can be used as an experimental model for the        disorder.    -   c. Binding of a candidate to cells or tissues or organs in vivo        can be detected using the tissues collected from the animals        treated with the candidate alone. The binding candidates usable        in the present disclosure can be also used for in vivo imaging,        wherein for example a selective candidate labeled with a        detectable moiety is administered to a human or an animal,        preferably into the bloodstream, and the presence and location        of the labeled candidate in the host is detected. The candidate        can be labeled with any moiety that is detectable in human        and/or animals whether by nuclear magnetic resonance, radiology,        fluorescence, or other detection means known in the art.    -   d. Combination of in vivo and in vitro methods of candidate        binding and detection as mentioned above can be also used        preferably in animals. For example, a selected candidate labeled        with a moiety can be administered to an animal, the candidate        can bind to or interact with its target in vivo, followed by        scarification of the animal, collection of tissue or organ        samples and detection of the bound candidate in vitro using        detection means known in the art.    -   e. Embryo culture system An organism embryo (i.e., chicken or        zebra fish) culture system can be used to detect the inhibitory        effect of candidates against pathogens (i.e., viruses). A middle        term embryo is treated with at least one selected candidate or        one combination of at least two selected candidates at a low        dosage within the range that yields efficacy of blocking or        acting without much extras, in the ordinary skill in the art;        pathogen inoculation can be performed next day. Candidates can        be also administrated one day after pathogen inoculation. Rest        of experiments including harvesting fluid containing pathogens,        determining pathogen titers can be performed in the ordinary        skill in the art. Lower pathogen tiers from an embryo culture        being treated with a candidate or one combination of at least        two candidates compared to controls without candidate treating        can indicate an efficacy of prevention or therapy.

Other cellular or tissue culture and animal experiments depending onneeds are also included.

Features or Characteristics of Therapeutic Targets

Based on the results of cellular or tissue culture and animalexperiments as described above, the features or characteristics andutilities of therapeutic targets include but not limited to thefollowing:

Type A: A therapeutic target is the binding site of a pathogen but not apathogenic site (the therapeutic target does not induce significantbiological disorders in an organism).

This therapeutic target can be the receptor of the antibodies induced byan effective vaccine and an effective passive immunity withoutsignificant side effects, and should be the goal of vaccine developmentand antibody prevention and therapy. The therapeutic target is also agood candidate of immunogen for a binding-site vaccine. The agonists andother derivatives of the therapeutic target are good candidates for theprevention, treatment and drug delivery tools of the diseases relevantto the target (i.e., a viral infection).

A candidate capable of binding to the therapeutic target as describedabove can be directly used as drugs for prevention, therapy and drugdelivery tool of the relevant disorder.

Type B: A therapeutic target is the binding site of a pathogen and apathogenic site (the therapeutic target induces significant biologicaldisorders in an organism).

This type of therapeutic target can be the receptor of the antibodiesinduced by an effective vaccine and an effective passive immunity withsignificant side effects. The vaccine and antibody prevention andtherapy should be administrated carefully. That is, efficacy for theblocking of pathogen binding sites should be sought while trying tominimize any extra free antibodies. The antagonists and otherderivatives of the therapeutic target are good candidates for theprevention, treatment and drug delivery tools of the diseases relevantto the antigen (i.e., a viral infection).

Candidates capable of binding to the therapeutic target as describedabove can be the causes of inflammation, autoimmune diseases, allergy,cancers, obesity and other disorders. These candidates can be used asdrugs for diagnosis of these disorders, therapy and drug delivery toolof the relevant disorder either at low dose or conjugated with anantagonist or inhibitor of the therapeutic target. These candidates canalso be used for development of animal models of the relevant disorders.

Type C: A therapeutic target is not the binding site of a pathogen but apathogenic site (induces significant side effects, toxicity ordisorders).

This type of therapeutic target should be avoided in vaccinedevelopment. Candidates capable of binding to this type of therapeutictarget can be the causes of bio toxicity, pathogenic mechanisms ofdiseases with high death rates (i.e., avian influenza and Ebola virusinfection), autoimmune diseases, allergy, cancers, inflammation, obesityand other disorders. These candidates can be used for diagnosis of thesedisorders, and should be avoided in direct use for the treatment of therelevant disorders. However, these candidates can be used as drugs fortherapy and as drug delivery tools of the relevant disorders byconjugation with an antagonist or inhibitor of the therapeutic target.On the other hand, these candidates can be used for development ofanimal models of the relevant disorders.

Type D: A target is neither the binding site of a pathogen nor apathogenic site. This type of therapeutic target can be the cause of avaccine's ineffectiveness and should be avoided in vaccine development.

Candidates capable of binding to this type of therapeutic targets shouldbe avoided for all applications in an organism except as a drug deliverytool if such a binding is organ and/or tissue specific.

Binding site-based new therapeutics for prevention and treatment of aninfection can be achieved based on following mechanisms: 1) to competewith the pathogen binding site. 2) to block the pathogen binding site.3) to modify the chemical nature of the pathogen binding site.

Pathogenic site-based new therapeutics for prevention and treatment ofinfections, cancers, autoimmune diseases, allergy, inflammation, obesityand other disorders can be achieved based on following mechanisms: 1) toinhibit the pathogenic site; 2) to neutralize or compete with thepathogenic, inducer; and 3) to modify the chemical nature of thepathogenic site.

Numerous other features or characteristics of therapeutic targets canbecome readily apparent from the detailed description.

Utilities of Glycan-Based Array

Glycan-based array and the therapeutic targets identified by aglycan-based array according to the present disclosure have severalutilities as described herein, including utility suitable for humans,animals, plants and other organisms, such as: 1) The rapididentification of therapeutic targets; 2) The pathogenesis studies andcause screening of infectious diseases, autoimmune diseases, allergies,toxicity, cancers, obesity and other disorders; 3) The development ofanimal models of autoimmune diseases, allergies, toxicity, cancers,obesity and other disorders; 4) The development of high quality and newvaccines (i.e., binding site-based vaccines); 5) The effective controlof pandemic diseases (i.e., binding site-based prevention and therapy);6) The functional, toxic, pharmacological and pharmaceutical studies ofplant lectins, herbs, toxins and small molecules; 7) The rapid discoveryof drugs and drug delivery systems relevant to therapeutic targets; 8)The application of therapeutic targets, their derivatives and any otherforms of the therapeutic targets for the diagnosis, prevention,treatment and drug delivery of infectious diseases, autoimmune diseases,allergies, cancers, obesity and other disorders related to at least onetherapeutic target; 9) The studies of epidemiology and biologyespecially evolutionary biology; and 10) Numerous other utilities ofmolecular mimicry array can become readily apparent from the detaileddescription in current disclosure and PCT/US2007/018258.

Glycan-Related Therapeutic Targets and Infectious Diseases

In one embodiment of the present disclosure, a useful tool is providedfor understanding the etiology, pathogenesis, treatment, and preventionof infectious diseases as described in PCT/US2007/018258.

In addition, small animal models for HIV infection can be developedbased on the new organ tropism of HIV (i.e., mouse intestine). Smallanimals include but not limited to mouse, rat, guinea pig, rabbit, etc.Animals are infected with either viable or inactivated HIV followed bydetection of HIV in the tropic organs (i.e., mouse intestine).

Glycan-Related Therapeutic Targets and Inflammation, AutoimmuneDisorders and Allergies

In another embodiment of the present disclosure, a useful tool isprovided for understanding the etiology, pathogenesis, treatment, andprevention of autoimmune disorders as described in PCT/US2007/018258.

In addition, antibodies and other candidates as described in the currentdisclosure can be also used for diagnosis, prevention and treatment ofinflammation, cancers, autoimmune disorders and allergies with thesimilar methods as described in PCT/US2007/018258.

Glycan-Related Therapeutic Targets and Cancers, Obesity, and OtherDisorders

In yet another embodiment of the present disclosure, a useful tool isprovided for understanding the etiology, pathogenesis, treatment, andprevention of cancers, obesity, and other disorders as described inPCT/US2007/018258.

In addition, antibodies and other candidates as described in the currentdisclosure can be also used for diagnosis, prevention and treatment ofinflammation, autoimmune disorders and allergies and other disordersusing the similar methods as described in PCT/US2007/018258.

The Mechanisms of Vaccination and Passive Immunity and New Vaccines

In another embodiment of the present disclosure, a useful tool isprovided for understanding the mechanisms of vaccination and passiveimmunity and development of new and high quality vaccines as describedin PCT/US2007/018258.

In addition, the candidates as described in the current disclosure canbe used to develop binding-site vaccines as well as anti-multi-pathogenvaccines. Evaluation of the binding site features of the antibodiesinduced by a vaccine is critically important to judge whether a vaccineis good or not.

Therapeutic Target-Based Prevention and Treatment

The present disclosure also extends to a strategy for developing novelmethods of prevention, diagnosis, and treatment of the relevantdisorders, obtainable based on the therapeutic target being identified,including, but not limited to methods suitable for humans, animals,plants and other organisms.

As described in PCT/US2007/018258 and the current disclosure, candidates(including antibodies) related to therapeutic targets existing inorganisms including humans or animals can be used for the diagnosis,prevention (therapeutic target-based prevention) and treatment(therapeutic target-based therapy) of infectious diseases, autoimmunedisorders, allergies, cancers, inflammation, obesity, and otherdisorders. In addition, such candidates can be also used as drugdelivery tools for treatment of infectious diseases, autoimmunedisorders, allergies, cancers, inflammation, obesity, and otherdisorders.

Another strategy would be to use the derivatives of therapeutic targetsfor the diagnosis, prevention and treatment of infectious diseases,autoimmune disorders, allergies, cancers, inflammation, obesity, andother disorders using the similar methods as described inPCT/US2007/018258 and the current disclosure.

In addition, derivatives of therapeutic targets can be also used as drugdelivery tools for treatment of infectious diseases, autoimmunedisorders, allergies, cancers, inflammation, obesity, and otherdisorders.

Another subject of the present disclosure is the use of inactivatedparticles or fragments or extracts of a pathogenic reagent which sharesa therapeutic target with a biological organism for the prevention andtreatment of the related infectious diseases, autoimmune disorders,allergies, cancers, inflammation, obesity, and other disorders asdescribed PCT/US2007/018258 and the current disclosure.

In addition, inactivated particles or fragments or extracts of apathogen can be also used as drug delivery tools for treatment ofinfectious diseases, autoimmune disorders, allergies, cancers,inflammation, obesity, and other disorders.

Another subject of the present disclosure is the use of a pathogenicallytherapeutic target and/or its relevant candidates and/or its derivativeswhich causes an autoimmune disease for diagnosis of the autoimmunedisease, cancers and other related disorders. Kits containing pathogensor therapeutic targets obtained through the process of the presentdisclosure and/or antibodies to the pathogens or therapeutic targets canbe prepared in a variety of ways well known to those of ordinary skillin the art. Such kits are used to detect the presence of the antibody tothe antigen in a biological sample.

According to the present disclosure, pharmaceutically usefulcompositions comprising the therapeutic targets and/or its derivativesor any other relevant candidates of the therapeutic target can beformulated, according to known methods such as by the admixture of apharmaceutically acceptable carrier. Such compositions can contain aneffective amount of the antigens or other forms of the antigen and/orits relevant candidates or any other relevant candidates of thetherapeutic target to form a pharmaceutically acceptable compositionsuitable for effective administration.

The dosage regimen utilizing the therapeutic targets and/or itsderivatives or any other relevant candidates of the therapeutic targetaccording to the present disclosure is selected in accordance with avariety of factors including location and density of the antigen, type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularsubstances thereof employed. Optimal precision in achievingconcentrations of the said substances of the present disclosure withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the thereof employed substance availability to targetsites. This involves a consideration of the distribution, equilibrium,and elimination of the thereof employed substances of the presentdisclosure.

The present disclosure also has in one embodiment the objective ofproviding suitable topical, oral systemic and parenteral pharmaceuticalformulations for use in the novel methods of prevention and treatment.The compositions containing the therapeutic targets and/or its relevantcandidates and/or its derivatives or any other forms of the therapeutictarget identified as the active ingredient can be administered in a widevariety of therapeutic dosage forms in conventional vehicles foradministration. For example, therapeutic targets and/or its relevantcandidates and/or its derivatives or any other forms of the therapeutictargets can be administered in such oral dosage forms as tablets,capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, nasal drops, an injectable, aninfusion, or a form conjugated to a nano-particle.

The pharmaceutical compositions can be provided to a biological organismby a variety of routes such as subcutaneous, topical with or withoutocclusion, oral, intramuscular, intravenously (both bolus and infusion),intraperitoneally, intramuscularly, subcutaneously, intracavity, ortransdermally, inhalant, or other using forms well known to those ofordinary skill in the pharmaceutical arts.

A Formulation with Glycans, Lectins or Herbs and Small Molecules

Another subject of the present disclosure is a binding site-based drugformulation for the prevention or treatment of an infectious disease, acancer, an autoimmune disease, an allergy, inflammation, atoxin-relevant biological injury or another disease in a target host.The drug formulation comprising at least one of the identified detectioncandidates or the identified target candidates, and derivatives thereof,The drug formulation consisted of at least one of the identifieddetection candidates or the identified target candidates, andderivatives thereof: 1) glycans including sialic acids and hydroxylsubstituents thereof which has an identical or similar three dimensionalstructure to a glycan-related pathogen binding site or a glycanrelatedtherapeutic target: to compete with the pathogen binding site or thetherapeutic target; 2) lectins including but not limited to plantlectins or herbs or antibodies which can bind to a glycan-relatedpathogen binding site or a glycan-related therapeutic target: to blockthe pathogen binding site or the therapeutic target; and 3) smallmolecules including but not limited to sulfur containing compounds orproducts: to modify the chemical nature of the glycan-related pathogenbinding site or the glycan-related therapeutic target.

Glycans, lectins, herbs, antibodies and small molecules are as describedabove.

Sulfur containing compounds or products include inorganic and organiccompounds of sulfur. Inorganic compounds of sulfur include but notlimited to sulfate (SO₄ ²⁻), salts of sulfuric acid.

Organic compounds or products of sulfur include but not limited to asulfonate, a sulfonyl, a sulurate, a sulfide, and a sulfur containingamino acid. The general formula of sulfonate is RSO₂O⁻., where R is someorganic group. They are conjugate bases of sulfonic acids with formulaRSO₂OH. As is common, the same term is used for compounds containingthis functional group, ionic salts, or similar covalent compounds,esters. Sulfur containing compounds or products also include garlicproducts including but not limited to garlic powder, garlic oil andextract of garlic (Allicin, Allium sativum, Ajoene, etc.).

A sulfonyl group is an organic radical or functional group obtained froma sulfonic acid by the removal of the hydroxyl group. Sulfonyl groupscan be written as having the general formula R—S(═O)₂—R′, where thereare two double bonds between the sulfur and oxygen. The names ofsulfonyl groups typically end in-syl, such as in tosyl chloride which isp-toluenesulfonyl chloride, CH₃C₆H₄SO₂Cl or mesyl chloride which ismethylsulfonyl chloride,

CH₃SO₂Cl. Sulfonyl groups can be reduced to the hydrocarbon with lithiumaluminium hydride (LiAlH₄).

An Anti-Infectious Pathogen Fabric or Surface

Another subject of the present disclosure is an anti-infectious pathogenfabric or anti-infectious pathogen surface will be created by the stepof incorporating at least one of the identified detection candidates orthe identified target candidates into or on fibers; or providing atleast one of the identified detection candidates or the identifiedtarget candidates on supporting materials and making it into particles,and then attaching the particles onto a surface including but notlimited to a cloth, a mask, a cap, or goggles.

Therapeutic Application of Sialic Acids

Sialic acid (Sia) is a generic term for the N- or O-substitutedderivatives of neuraminic acid, a nine-carbon monosaccharide. Members ofthis group include: N-acetylneuraminic acid (Neu5Ac or NANA),2-Keto-3-deoxynononic acid (Kdn), N-Acetylglucosamine (GlcNAc),N-Acetylgalactosamine (GalNAc), N-Acetylmannosamine (ManNAc), andN-Glycolylneur-aminic acid (Neu5Gc). The amino group bears either anacetyl or a glycolyl group. The hydroxyl substituents may varyconsiderably: acetyl, lactyl, methyl, sulfate and phosphate groups havebeen found. In addition, the small molecules in the present disclosurealso include glycan binding molecules including but not limited toacetyl-, methyl- and sulfur-containing molecules as donors foracetylation, methylation and sulfatation of sialic acids or otherglycans. Glycan-binding antibodies are also included. The products ofsialic acids include substances containing at least one molecule ofsialic acids and derivatives of sialic acids. Glycolipids (e.g.Ganglioside) are also included.

a. Blocking Infection Through Competing with Pathogen Binding Sites

In one embodiment of the invention, a sialic acid is used as an agonistdrugs by competing with natural virus binding sites (or blocking) ortoxins. The application of N-acetylneuraminic acid (Neu5Ac) for theprevention and treatment of viral infections of rotavirus, influenza Aviruses H1N1, 2009 H1N1, H3N2 and H5N1, as well as allergy.

b. Modification of Sialic Acids

In one embodiment of the present invention, a sialic acid is modifiedusing acetyl-, methyl- and sulfur-containing molecules as donors (drugs)for acetylation, methylation and sulfatation of sialic acids or otherglycans. For example, methionine contains —S—CH₃ thus can be used as adonor to modify a pathogen binding site (a sialic acid or a glycan) intomethylated and sulfated forms. Sulfur- and containing compounds such assulfides (e.g. garlic products) as described in PCT/US2009/039810 canalso act as donors of CH₃—S—S—CH₃. Such chemical modification of asialic acid can attenuate even prevent pathogen binding to it.

The application of methionine, methionine-Zinc (Zn) complex and aformula consisted of Acetylneuraminic acid and methionine ormethionine-Zn for the prevention and treatment of rotavirus infection;N-Acetylneuraminic acid, N-Acetylneuraminic acid methyl ester and aformula consisted of Acetylneuraminic acid methyl ester for thetreatment of influenza infection are described in Exemplification (Table4 and 5). The application of a garlic product for the prevention andtreatment of viral infections of rotavirus, influenza A viruses H1N1,Newcastle disease viral (NDV), as well as allergy are described inPCT/US2009/039810. Such application of chemical molecules can beextended to other infections of other pathogens using sialic acids orother glycans as their binding sites.

c. Mis-Targeting of Microbial Sialidase

In another embodiment of the present invention, free sialic acids isused as false-targets, bind to and mis-activate (mis-lead) microbial orhost sialidase. The mis-targeting protects host cells from beingattacked by microbial sialidase, or protect the Sia-shedding on thesurface of host cells. Further, microbial sialidase can be exhaustedthrough inefficient work. This mechanism is different from thecompetitive inhibitors of sialidase. Metals can bind or conjugate tomicrobial sialidase and cell-surface (plasma membrane) and cytoplasmicsialidases, interrupt or inactivate sialidase function.

The application of N-acetylneuraminic acid (Neu5Ac) for the treatment ofviral infections of rotavirus, influenza A viruses H1N1, 2009 H1N1, H3N2and H5N1, as well as allergy are described in Exemplification.

d. Neutralizing Sialic Acid-Binding Molecules

In one embodiment of the invention, sialic acids or the derivatives ofsialic acids are used to bind to and neutralize or modifyingglycan-binding molecules of harmful toxins, lectins and antibodies andthus protect host cells.

One evidence supporting this mechanism of action is that after beingtreated (neutralization) with Neu5Ac, the harmful anti-rotavirusantibodies mentioned in the exemplification did not induce mouse deathswith rotavirus infection (Table 3).

Another supporting evidence is the efficacy of Neu5Ac andN-acetylneuraminic acid methyl ester for the treatment of the sideeffect of influenza antibodies. As described in exemplification,injection with moderate or high dose of antibodies against 2009H1N1(swine), seasonal H1N1 and avian H5N1 influenza virus into day 15-16 or18-19 chicken embryo induced either deaths or the leg disability ofnewborn chicks which is similar to the Guillain-Barre syndrome (GBS) inhuman (FIG. 14). However, newborn chicks with injection of the sameantibodies pre-treated with N-acetylneuraminic acid (Neu5Ac) did notdevelop the syndrome.

New Vaccines and the Acting Mechanism of Antibody Therapy

In vivo proof of a novel action mechanism of vaccination and antibodies

In vitro proof of a novel action mechanism of vaccination, passiveimmunity and antibody therapy has been described in PCT/US2007/018258.The functional mechanism is that antibodies induced by an infection or avaccine or acquired through passive immunity bind to and block at leastone binding site of a pathogen.

One example is that goat IgG in sera of pups delivered to those damsinjected with goat-anti-rotavirus antibodies were undetectable at theday of birth. At the same time, binding of goat IgG to goblet cells ofsmall intestine of the pups were detected at age of week 1 (FIG. 7)through week 3. In addition, oral administration of low dose of the sameanti-rotavirus antibody showed efficacy for the prevention and treatmentof rotavirus infection in a mouse model (PCT/US2007/018258).

Similarly, binding of goat IgG to the lung of the mouse pups deliveredto the dams injected with goat anti-respiratory syncytial virus (RSV)antibodies was detected up to age P9 (FIG. 8) and P14. Usinganti-respiratory syncytial virus (RSV) antibodies such as Palivizumab(Synagis®) for protecting premature infants from severe RSV disease hasshown efficacy. However, the mechanism of action of the drug is unclear.The mechanism of action disclosed in the present invention could be thefunctional mechanism of Palivizumab.

For further in vivo proof, day 16 (E16) chicken embryos were treated viaallantois injection of anti-influenza virus immune sera; the blood werecollected from the newborn chickens at day 3 after birth (day 8 afterserum injection); then the chicks were infected with the 2009H1N1(California) virus. The newborn chicks with injection of the seracontaining anti-2009H1N1 (California) and seasonal H1N1 (Shanghai, 1999)viruses were not infected by the 2009H1N1 virus (Table 11). The antibodylevels in the sera collected before viral infection were zero determinedby an inhibitory hemagglutination test. Thus the action mechanism of theantibodies mentioned above was blocking of viral binding sites ratherthan neutralization of virus as described in exemplification.

Anti-Pathogen Antibodies as Drugs for Antibody Prevention and Therapy

Anti-pathogen antibodies as drugs for antibody prevention and therapyhave been described in PCT/US2007/018258. The present invention providesfurther in vivo proof.

As described above and in exemplification (Table 5, 6, 7, 10 and 11),low dose of following immune sera or antibodies can be used as drugs forantibody-prevention and antibody-therapy of influenza infections.

-   -   a. Anti-seasonal H1N1 virus antibodies for the prevention of the        2009H1N1 (swine), other H1N1 and H5N1 influenza virus infection;    -   b. Anti-2009H1N1 (California) virus antibodies for the        prevention of the 2009H1N1 (swine), other H1N1 and H5N1        influenza virus infection; and    -   c. Anti-H5N1 virus antibodies for the prevention of the 2009H1N1        (swine), other H1N1 and H5N1 influenza virus infection.        The best efficacy of antibody-prevention and antibody-therapy        for influenza infections was achieved using low dose of        antibodies pre-treated with Neu5Ac or Neu5Ac-methyl ester as        described in exemplification (Table 5, 6, 7, 10 and 11, FIG.        14D).

Anti-Multiple Pathogen Vaccines for Influenza Infections

As described in exemplification, the anti-2009H1N1 (California) andanti-H5N1 immune sera (pre-treated with Neu5Ac) were effective forprevention and treatment of the A/PR/8/34(H1N1) virus infection in mousepups (Table 5 and 6); the anti-H1N1 (seasonal) human sera and anti-H5N1immune serum (pre-treated with Neu5Ac) were effective for the preventionand treatment of the 2009H1N1 (California) virus infection in chickenembryos and newborn chicks (Table 7 and 10). These results provide invivo evidence that influenza viruses of 2009H1N1 (California), seasonalH1N1 and avian H5N1 share at least one binding site. Thus one embodimentof the present invention is disclosing following new vaccines based onthe shared binding sites.

-   -   a. A seasonal H1N1 virus vaccine for the prevention of the        2009H1N1 (swine), other H1N1 and H5N1 influenza virus infection;    -   b. The 2009H1N1 (California) virus vaccine for the prevention of        the 2009H1N1 (swine), other H1N1 and H5N1 influenza virus        infection; and    -   c. A H5N1 virus vaccine for the prevention of the 2009H1N1        (swine), other H1N1 and H5N1 influenza virus infection.        Dualistic Roles of Antibodies

The roles of antibodies can be dualistic; they can act either asprotectors by recognition and blocking pathogen binging sites; ordisease inducers by triggering of certain harmful pathways as describein PCT/US2007/018258. The present invention provides further in vivoproof.

a. High Levels of Antibodies are Harmful

As described in exemplification (Tables 5-10), high dose of the seracontaining either anti-seasonal H1N1 antibodies or anti-2009H1N1antibodies was not effective or harmful for the prevention of theA/PR/8/34(H1N1) infection of newborn pups. Further, high dose of thesera induced either death or severe disease in chicken fetus and newbornchicks (Table 7 and FIG. 13).

b. Guillain-Barre Syndrome (GBS)

Injection with moderate or high dose of the anti-H1N1 and anti-H5N1antibodies into E16 chicken embryo induced the leg disability of newbornchickens (FIGS. 13B, 13C and 13D) which is similar to the Guillain-Barresyndrome (GBS) in human. The antibodies induced by the 2009H1N1(California) virus is at highest risk for inducing GBS, followed byantibodies induced by the avian H5N1 (Anhui, 2005) virus and theseasonal-H1N1 (Shanghai, 1999) virus.

c. Pathogenic Mechanisms of Harmful Antibodies

Pathogenic mechanisms of harmful antibodies have been described inPCT/US2007/018258, PCT/US2009/039810 and U.S. 61/278,685. Otherpathogenic mechanisms include but not limited to antibody-dependentcytotoxicity.

Treatment of Harmful Antibodies

One embodiment the present invention discloses using the following drugsand drug candidates for developing the treatment of a severe lowrespiratory infection including but not limited to 2009 H1N1, H5N1 andRSV infections, other infectious diseases, autoimmune diseases, cancers,obesity and other disorders to neutralize, dilute or interrupt thebinding of harmful antibodies to their targets.

-   -   a. Sialic acids and derivatives including Ganglioside products;    -   b. Kinase inhibitors;    -   c. Beta-2 receptor inhibitors    -   d. Acetyl-, methyl- and sulfur-containing molecules as donors        for acetylation, methylation and sulfatation of sialic acids or        other glycans (modification and inactivation).    -   e. Immunoglobulin products or serum; and    -   f. Any other substances and approaches to interrupt kinase        activating pathways or the binding of harmful antibodies to        their targets.

Animal Models for Rapid Evaluation of Vaccines and Antibodies

One embodiment of the present invention is experimental models usingcellular culturing or animals and anti-pathogen antibodies forevaluating efficacy and safety or side effect of vaccines or antibodiesas described in exemplification. An experimental model for thepathogenesis study of Guillain-Barre syndrome (GBS) and evaluation ofside effect of influenza vaccines and antibodies is also described inexemplification (FIG. 12 and FIG. 13). These experimental models can beused but not limited for screening drugs for prevention and treatment ofGBS or for the rapid evaluation of the efficacy and safety of vaccinesand antibodies.

EXAMPLES 1. An Array Carrier

FIG. 1 shows an example of an array chip. A, B, C, D, E and F representdifferent kinds of target candidates attached to the chip; G representsan example of histochemistry staining; and H represents an example offluorescent staining.

2. Primary Screening of Potential Therapeutic Targets Using PlantLectins

FIG. 2 and FIG. 3 show examples of screening of potential biologicaltherapeutic targets using plant lectins as detection candidates. Plantlectins used were purchased from Vector Laboratories (California, USA)and included: wheat germ agglutinin (WGA) which specifically recognizesN-Acetyl-D-Glucosamine, Ulex Europaeus agglutinin I (UEA I) whichspecifically recognizes α-Focus, and soybean agglutinin (SBA) whichspecifically recognizes N-Acetyl-D-Galactosamine. All lectins werelabeled with biotin, and another kind of WGA was labeled with afluorescent (Rhodamine). The secondary reagent for biotin-labeledlectins was fluorescent (Texas Red) conjugated-streptavidin.

Biotin-labeled lectins WGA and SBA were incubated for one hourseparately with tissue sections of heart, lung, liver, kidney and spleenof a bulb/c adult mouse (FIG. 2); WGA and UEA I were incubatedseparately with tissue sections of small intestines of bulb/c newbornpups and adult mouse (FIG. 3); and WGA were incubated separately withtissue sections of liver and small intestine of bulb/c mouse pupsinfected or uninfected with rhesus rotavirus (RRV) (FIG. 4). After wash,streptavidin-Texas Red was added and incubated for 30 minutes followedby wash and detection with a fluorescent microscope. Positive binding isshown as areas stained brightly (white) and negative binding as areasnot stained brightly (grey areas).

As shown in FIG. 2 and indicated by binding of WGA,N-Acetyl-D-Glucosamine is expressed strongly on part but not all ofheart, lung; moderately on certain cells of spleen; and negatively onliver and kidney of bulb/c mice (FIG. 2A). Indicated by binding of SBA,NAcetyl-D-Galactosamine is expressed moderately on certain cells ofspleen and negatively on heart, lung, liver and kidney of bulb/c mice(FIG. 2B). As shown in FIG. 3 and indicated by binding of WGA and EUA I,N-Acetyl-D-Glucosamine (FIG. 3A) but not α-Focus (FIG. 3B) is expressedstronger on small intestine of day 2 and day 4 newborn mouse pups andweaker on small intestine of adult mice. Based on the primary screeningresults, N-Acetyl-D-Glucosamine and N-Acetyl-D-Galactosamine arepotential biological markers related to diseases. Whether they arebinding sites of a pathogen will be detected by binding of the pathogenand WGA or SBA to an array chips consisted of healthy tissue sections ofhumans, animals, and plants or various cell lines as illustrated above.

This process will be easily extended to bind other lectins specific forother glycans to the array chip mentioned above to detect otherglycan-related potential biological markers existing in humans, animalsand other organisms.

3. Identification of Glycan-Related Biological Markers

FIG. 4 shows an example of identification of a glycan-related biologicalmarker by binding of WGA to healthy and disease tissue sections.

Two groups of sucking bulb/c mouse pups were treated at day 2 afterbirth via oral administration with 30 μl of saline (uninfected group)and 30 μl of RRV at concentration of 1×10⁷ pfu/ml (RRV infected group).The course of this viral illness is that within week 1 after RRVinfection, the pups have diarrhea with alcoholic stools, don't eat welland fail to gain weight as quickly as healthy mice; and 30-40% of pupswith serious illness become jaundiced. By the 2nd week all the micebecome jaundiced, don't eat well and fail to gain weight; and 80% ofpups with serious illness died. Viruses are usually cleared within 5days and undetectable at day 5. Pups were sacrificed at different daysafter treatment and samples of sera, snap-frozen and formalin-fixedtissues of intestine and liver, were processed.

As shown in FIG. 4 and indicated by binding of WGA, the livers and smallintestines from mouse pups infected with RRV within one week (acutephase) were filtrated with inflammatory or proliferating cells withstrong expression of N-Acetyl-D-Glucosamine (FIG. 4B). The livers andsmall intestines from healthy pups without RRV infection are negativefor the glycan expression (FIG. 4A). Therefore, glycanN-Acetyl-D-Glucosamine is a potential biological marker related toinflammation.

In another experiment, WGA binding to human healthy and disease tissueswas detected with a tissue array chip consisted of FDA normal humanorgans (Array I+II)+bonus malignat samples (Immgenex, San Diego, USA).The results showed that majority of normal human organs except bonemarrow, salivary gland and hypophysis do not or weakly expressN-Acetyl-D-Glucosamine, while following cancer tissues strongly expressN-Acetyl-D-Glucosamine: malignant melanoma, brain malignantoligodendroglioma, kidney clear cell carcinoma, skin basal cellcarcinoma of head, throat carcinoma, Hodgkin's lymphoma ofsupraclavicular, colon intermediate grade interstitialoma, thyoidmedullary carcinoma and skin squamous cell carcinoma of left chest wall.

Similarly, other biological markers relating to cancers and otherdiseases can be easily identified by binding of WGA and other lectinsspecific for other glycans to an array chip consisted of healthy andcancer tissue or other disease tissue sections of humans and animals, orvarious tumor cell lines as illustrated above.

4. Identification of Potential Disease Inducers

FIG. 5 shows an example of identification of potential disease inducersby binding of an anti-rotavirus (RV) polyclonal antibody and lectin WGAto tissue sections of RRV infected and uninfected mouse pups. Thebiotin-labeled anti-RV polyclonal antibody was purchased from MeridianLife Science, Inc (Mine, USA). Briefly, the Rhodamine-labeled WGA andthe biotin-labeled anti-RV antibody were incubated for one hour withtissue sections of small intestines from the uninfected and infectedmouse pups as mentioned above. After wash, streptavidin-conjugatedhorseradish peroxidase (HRP) was added and incubated for 30 minutesfollowed by wash, HRP substrate DAB was added and incubated for 15minutes followed by was and detection with a regular and fluorescentmicroscope. Positive binding for the antibody is shown as areas stainedbrown, and positive binding for WGA is shown as areas stained brightlyred and negative binding as areas not stained brightly (dark areas).

As shown in FIG. 5, the anti-RV antibody binds to the same proliferatinggoblet cells expressing N-Acetyl-D-Glucosamine (FIG. 5B) in the smallintestine of RRV infected pups (day 5 after RRV infection). In an acuteviral infection, viruses are usually cleared within one week, andanti-virus antibodies are at elevated levels from week 1 and reach peaklevels at week 2 to week 3. Because anti-RV antibodies bind toproliferating cell expressing N-Acetyl-D-Glucosamine, these antibodiescan cause inflammation even after viral clearance. This inflammation inturn leads to a proliferative response of the host defense system, whichfurther exposes the glycan target. Thus, anti-RV antibodies can be aninflammatory inducer and a cause of autoimmune diseases of tissues ororgans expressing N-Acetyl-D-Glucosamine. If the proliferatinginflammation persists long an autoimmune diseases can be developed. Ifthe proliferating inflammation eventually leads to an uncontrollablecell growth, a cancer can be developed. For these reasons, anti-RVantibodies can be an inducer of autoimmune diseases and cancers. Thiswill be further detected by comparison of binding of anti-RV antibodiesand WGA to healthy and disease tissues sections attached on an arraychip.

Similarly, other potential disease inducers can be easily identified bybinding of other antibodies against other pathogens and WGA or otherlectins specific for other glycans to an array carrier consisted ofdisease tissue sections of humans and animals, or various tumor celllines as illustrated above.

5. A Drug Formulation for Prevention and Treatment of Viral Infectionsand Allergy

A drug formulation consisted of commercially available syntheticN-Acetylneuraminic acid (Neu5Ac) (JunKang Biotech Co., Ltd, Guangzhou,China) and garlic oil products (Nature's Bounty, INC, New York, USA) wastested for its efficacy on prevention and treatment of viral infectionsand allergy as described below.

5.1. Prevention of Rotavirus Viral Infection

Three groups of sucking bulb/c mouse pups were treated at day 1 afterbirth via oral administration with 1) 20 μl of saline (saline treatedcontrol group, n=21); 2) 20 μl of the garlic oil at the concentration of1-5 μg/g body weight; and 3) 20 μl of the formulation consisted of theNeu5Ac (0.5-2 μg/g body weight) and the garlic oil (1-5 μg/g) (drugtreated group, n=20), followed by challenging with RRV at day 2 asdescribed above. Mice were kept for 3 weeks after RRV infection. Pupspretreated with the garlic oil product alone or the formulationconsisted of the Neu5Ac and the garlic oil product were not infectedcompared to control pups pretreated with saline. Representative 2week-old pups pr-treated with saline and the formulation are shown inFIG. 6A, and the representative histology changes of small intestine ofday 4 pups are shown in FIGS. 6B (saline pretreated) and 6C (formula ordrug pretreated). The results with statistic analysis are concluded inTable 2.

TABLE 2 The results of efficacy test of a formulation consisted ofNeu5Ac and garlic oil Pathogen Saline Formulation Odd Ratio Test Subjector Disease *Effec Ineffec (%) Effec Ineffec (%) (OR) 95% Cl P valueBulb/c mice RRV 4 17 (81) 18 2 (10) 0.03 0.03~0.47 <0.0001 Chicken NDC 1 9 (90) 7 3 (30) 0.05 0.13~0.88 0.02 Chicken embroy H1N1  1** 10 (91) 91 (10) 0.01 0.02~0.71 0.0003 Human Flu 1 14 (93) 15  1 (6.3) 0.0050.01~0.45 <0.0001 Human Allergy 2 13 (87) 14  1 (6.7) 0.07 0.01~0.52<0.0001 *Effec = effective, Ineffec = ineffective; **Viral titer lowerthan 1:16 were counted as effective.

5.2 Prevention and Treatment of Newcastle Disease Viral (NDV) Infection

Two groups of SPF chickens (4 weeks, ˜2 kg) were pretreated via nasaldrop and oral administration with 2 ml of saline (saline treated controlgroup, n=10), or 2 ml of the formulation at the concentration of 1 mg/mlof each Neu5Ac and the garlic oil (drug treated group, n=10), followedby challenging with highly pathogenic NDV next day. 20 ml of saline orthe formulation were separately added to 200 ml of drinking water ofeach group once at day 3 after viral infection. The course of this viralillness is that within one week after NDV infection, the chickens havediarrhea, don't eat well, and more than 50% of chickens die. Thechickens were kept for 8 days after NDV infection.

At day 8 after NDV infection, 7/10 (70%) of chickens treated with salinewere died and another 2 chickens were sick with diarrhea and did noteat; 1/10 (10%) of chickens treated with the formulation was died andanother 2 were sick. The formulation reduced total death rate of the NDVinfection from 70% to 10% (Odd Ratio=0.05, 95% CI=0.02˜0.96, P=0.02). Asconcluded in Table 2, the formulation reduced total sick(death+sick=ineffective) rate of the NDV infection from 90% to 30%.

5.3 Prevention and Treatment of Influenza Viral Infection of ChickenEmbryos

Two groups of chicken embryos were pretreated via injection with 100 μlof saline (saline treated control group, n=12), or 100 μl of theformulation as mentioned above at the concentration of 0.5 mg/ml of eachNeu5Ac and the garlic oil (drug treated group, n=12), followed byinoculation of influenza viral stain H1N1 into the allantois next day.100 μl of saline or the formulation were separately injected into theallantois once everyday after viral inoculation and allantois fluid wascollected at 48 hours after viral inoculation and viral titers in thefluid were determined by a hemagglutination test.

The viral titers of 10/12 of chicken embryos treated with saline were1:256, and the viral titers of 9/12 of chicken eggs treated with salinewere below 1:16 that were counted as effective in Table 2. The chickenembryos with viral titer of zero in their allantois fluid (1 for salinetreatment and 2 for formula treatment) were not included in statisticanalysis in order to exclude the possible failure of viral inoculation.As concluded in Table 2, the formulation significantly inhibited H1N1infection of chicken embryos.

5.4 Treatment of Influenza of Humans

Two groups of human subjects with influenza were treated twice every dayfor 2-3 days via nasal drop and oral administration each time with 15 mlof saline (saline treated control group, n=15), or 15 ml of theformulation at the concentration of 1 mg/ml of each Neu5Ac and thegarlic oil product (drug treated group, n=16). Symptoms of influenzainclude fever, headache, tiredness, cough, sore throat, runny or stuffynose, body aches, or diarrhea. The illness course is usually one week.The subjects were observed everyday for symptoms of influenza for 7days. Compared to the subjects treated with saline, 15 subjects treatedwith the formulation had significantly reduced symptoms and shorterillness course (5-7 days versus 3-4 days). Those subjects were countedeffective in Table 2.

5.5 Treatment of Allergy of Humans

Two groups of human subjects with seasonal allergy were treated everyday for 2-3 days via nasal drop each time with 1-2 ml of saline (salinetreated control group, n=15), or 1-2 ml of the formulation at theconcentration of 1 mg/ml of each Neu5Ac and the garlic oil (drug treatedgroup, n=15). Symptoms of allergy include nasal congestion, runny nose,sneezing and itchy nose. The subjects were observed everyday forsymptoms of allergy for 3 days. Compared to the subjects treated withsaline, 14 subjects treated with the formulation had significantlyreduced allergic symptoms. Those subjects were counted effective inTable 2.

6 Drug Candidates for Prevention and Treatment of Viral Infections

The following drug candidates were tested for its efficacy on preventionand treatment of viral infections and allergy as described below.

-   -   a. Synthetic N-Acetylneuraminic acid (Neu5Ac) (JunKang Biotech        Co., Ltd, Guangzhou, China).        -   Formula: C₁₁H₁₉NO₉.        -   Molecular weight: 309.3.        -   Purity (HPLC): 98.7%.

-   -   b. Methionine (J. R. Carlson Laboratories, Inc., Arlington Hts,        Ill., USA).        -   Formula: C₅H₁₁NO₅S        -   Molecular weight: 149.21

-   -   c. A garlic oil product contains 10 mg of allium sativum per        softgel (Nature's Bounty, INC, New York, USA). The effective        component is mainly diallyl disulfide.        -   Formula: C₆H₁₀S₂        -   Molecular weight: 146.28

-   -   d. A purified goat polyclonal antibody against the bovine        rotavirus strain NCDV (Nebraska Calf Diarrhea Virus) (Meridian        Life Science, Inc., Saco, Me., USA).        -   Purity: >95%        -   Concentration: 5 mg/ml.    -   e. A monoclonal antibody reacts with P41 major capsid protein        (VP6) of bovine and human rotavirus isolates (Meridian Life        Science, Inc., Saco, Me., USA).        -   Purity: >95%        -   Concentration: 0.1 mg/ml.    -   f. Diethyl disulfide (Lida Chemicals, Shijiazhuang, China).        -   Formula: C₄H₁₀S₂        -   Structure: CH₃CH₂SSH₂CH₃    -   g. Methionine-Zinc (Zn) complex (Jiande Biotech, Zhejiang,        China)        -   Zinc (Zn): 25%.    -   h. Synthetic N-Acetylneuraminic acid methyl ester (JunKang        Biotech Co., Ltd, Guang-zhou, China).        -   Molecular weight: 323.3        -   Formula: C₁₂H₂₁NO₉        -   Structure: See right

5.7 Prevention and Treatment of Rotavirus Viral Infection withFormulations

Four groups of sucking bulb/c mouse pups were treated at day 1 afterbirth (P1) via oral administration with saline (control group) or drugcandidate followed by challenging at day 2 (P2) with 20 μl (microliter)of rhesus rotavirus (RRV) at the concentration of 1×10⁷ PFU/ml. Thedifferent groups of mice were treated with each 20 μl (microliter)of: 1) saline (RRV group, n=11); 2) Neu5Ac at the concentration of 2mg/ml (Sia group, n=13); 3) methionine at the concentration of 2 mg/ml(Met group, n=12); and 4) a formulation consisted of the Neu5Ac (2mg/ml) and the methionine (2 mg/ml) (formula-1, n=12). Mice were keptfor 3 weeks after RRV infection.

TABLE 3 The results of efficacy test of drug candidates for rotavirus(RRV) infection Not Sick Odds P Drug candidate Sick sick Rate(%) Ratio95% CI value Saline 9 2 81.8 4.5 0.10-0.79 0.01 Neu5Ac 3 10 23.1 0.070.01-0.49 0.01 Metnionine 3 9 25.0 0.07 0.01-0.56 0.01 Neu5Ac + Meth* 210 16.7 0.01 0.01-0.38 0.003 *Meth = Metnionine.

The course of rotavirus infection is that within week 1 after RRVinfection, the pups have diarrhea with alcoholic stools, don't eat welland fail to gain weight as quickly as healthy mice; and some pups withserious illness become jaundiced. By the 2nd week all the mice becomejaundiced, don't eat well and fail to gain weight; and 80% of pups withserious illness died. Viruses are usually cleared and undetectablewithin one week.

Pups pretreated with the drug candidates were not infected compared tocontrol pups pretreated with saline. The results with statistic analysisare concluded in Table 3. The effective dosages of drug candidates forrotavirus infection for human are calculated and listed in Table 4.

TABLE 4 The dosages of drug candidates for rotavirus infection Drugcandidate Low (mg/kg) High (mg/kg) Range (mg/kg) Neu5Ac 0.1 20 0.1-20Metnionine 0.1 20 0.1-20 Neu5Ac + Meth* 0.1:0.1 20:20 0.1:0.1-20:20 

5.8. Prevention and Treatment of Rotavirus Infection with Low Dosage ofAntibodies

Five groups of bulb/c pups were treated at P0 via oral administrationwith each 20 μl (microliter) of saline solution containing differentdrug candidates followed by oral inoculation of 20 μl (microliter) ofRRV (1×10⁷ PFU/ml) at P1. The different treatments include: 1) salinealone (RRV alone, n=11); 2) the anti-NCDV antibodies (10 μg) (Ab-1+RRV,n=15); 3) the anti-VP6 monoclonal antibody (1 μg) (Ab-2+RRV, n=12); 4) acombination of the anti-NCDV (10 μg) and the anti-VP6 (1 μg) antibodies(Ab-1 & Ab-2+RRV, n=8); and 5) a formulation consisted of the anti-NCDVantibodies (50 μg) and the Neu5Ac (25 μg) (formula-2, n=6). Mice werekept for 3 weeks after RRV infection.

TABLE 5 The results of efficacy test of antibody therapy for rotavirusinfection Not Sick Odds P Drug candidate Sick sick Rate(%) Ratio 95% CIvalue Saline 9 2 81.8 4.5  0.10-0.79 0.01 Ab-1* 5 10 33.3 0.11 0.02-0.72 0.02 Ab-2** 2 10 16.7 0.04 0.005-0.38 0.003 Ab-1 + Ab-2 1 712.5 0.03 0.002-0.43 0.005 Ab-1 + Neu5Ac 1 5 16.7 0.04 0.003-0.62 0.02*anti-NCDV antibody; **anti-RV-VP6 antibody.

The course of rotavirus infection is as described above. The resultswith statistic analysis are concluded in Table 5.

In combination with the results of PCT/US2007/018258, the efficacy datashowed that 1) low dosage of the anti-NCDV antibodies (less than 20μg/each pup) reduced the severity and shorten the course of rotavirusinfection; 2) the anti-VP6 antibody alone or in combination with theanti-NCDV antibodies prevented rotavirus infection; and 3) Neu5Acreduced the toxicity (see below) of the anti-NCDV antibodies.

5.9 Prevention and Treatment of Influenza Infection of Chicken Embryos

Four groups of chicken embryos were pretreated via injection with 100 μlof saline solution containing 1) saline alone (control group, n=10); 2)50 μg of the Neu5Ac (Neu5Ac group, n=10); 3) 40 μg of the allium sativum(garlic group, n=5); and 4) 40 μg of the Neu5Ac and 40 μg of the alliumsativum (formula-2 group, n=5), followed by inoculation of the 2009 H1N1(swine) influenza viral stain (California) into the allantois next day.Another two groups of chicken embryos with the same treatment withsaline (control) and 50 μg of the Neu5Ac were inoculated with seasonalH1N1 (China) virus. 100 μl of saline or the Neu5Ac solution wereseparately injected into the allantois once everyday after viralinoculation and allantois fluid was collected at 48 hours after viralinoculation and viral titers in the fluid were determined by ahemagglutination test.

The viral titers of 3/5 of chicken eggs treated with the garlic were1:16 or 1:32; and the viral titers of 3/5 of chicken eggs treated withthe formula-2 (Neu5Ac+garlic) were 1:1 or 1:2. The results aresummarized below.

Group 1 3 4 Treatment Saline Garlic Formula-2 Viral titer 1:256 1:16-321:1-2

5.10 Treatment of Side Effect of Avian (H5N1) Influenza Vaccine withNeu5Ac

SPF chickens (4 weeks, ˜2 kg) were sick after 3 weeks of the treatmentwith a H5N1 vaccine candidate. Sick chickens looked tired and did noteat well. Two groups of the sick chickens were treated once a day viaoral administration with 2 ml of saline (control group, n=7), or 2 ml ofthe Neu5Ac at the concentration of 2 mg/ml (drug treated group, n=8),for three days. At the first day of treatment, chickens with Neu5Actreatment looked and ate better. At day 3, 7/8 of the sick chickensrecovered and looked normal while 6/7 of the saline-treated controlgroup still sick. The Neu5Ac reduced the side effect of the H5N1 vaccinecandidate from 87.5% to 12.5% (Odd Ratio=0.02, 95% CI=0.001˜0.40,P=0.01).

6. Pathogenic Antibodies

Antibodies can be harmful if they bind to a molecule linking to aharmful activating pathway such as kinase pathways.

6.1 Antibodies from Pregnant Mother Induced Disease

Pregnant bulb/c mouse dams were treated via intraperitoneal injectionwith 250 μg of the anti-NCDV antibodies three times at E14, E16 and E18before delivery without RRV inoculation to pups (n=21). 7 (33.3%) of thepups delivered to those dams were died at birth; and 52.4% of the pupsfailed to gain weight from day 10 after birth, and developed an illnessthat was similar to experimental biliary atresia (obstruction of bileduct). The bile duct and gall bladder of the pups looked twisted at week2 when compared to the bile duct and gall bladder of control pupsdelivered to the dams treated with saline (FIG. 9A). Further,significant proliferation of bile duct epithelium cells (FIG. 9B) andinfiltration by inflammatory cells in liver (FIG. 9C) was observed atweek 1 in those pups (FIG. 10: Ab to mother). In another experiment,injection of 15 μg of anti-RV antibody once at E18 caused significantinflammatory proliferation of bile duct epithelium cells at P4 and thoseproliferating cells blocked the bile duct from P5 (FIG. 9E) compared tonormal control pups (FIG. 9D).

6.2 Antibodies Plus RRV Induced Severe Viral Infection

Four groups of bulb/c pups were treated via intraperitoneal injectionwith each 20 μl (microliter) of 1) saline at P0 and RRV at P1 (controlgroup, n=21); 2) the anti-NCDV antibodies (50 μg) at P0 and RRV at P1(Ab+RRV, n=14); 3) RRV at P0 and antibodies (50 μg) at P1 (RRV+Ab,n=10); 4) Antibodies (75 μg) to pregnant mouse at pregnant day 18 (E18)and RRV to pups at P0 (Ab to mom+Ab, n=21); and 5) 250 μg of theanti-NCDV antibodies three times at E14, E16 and E18 before deliverywithout RRV inoculation to pups as mentioned above. The RRVconcentration was 1×10⁷ PFU/ml. Mice were kept for 3 weeks after RRVinfection.

TABLE 6 The results of high dose of antibodies in rotavirus infectionTreatment Pup number Death Sick Death (%) Sick (%) Saline + RRV 28 9 2432.1 85.7 *Ab (pup) + RRV 14 4 11 28.6 78.6 RRV + Ab (pup) 10 1 6 10.060.0 Ab (mom) + RRV 21 10 20 47.6 47.6 Ab to mom 21 7 11 33.3 52.4 *Ab =anti-NCDV antibody

The course of rotavirus infection is as described above. The results aresummarized in Table 6 and FIG. 10.

As shown in FIG. 10, the pups delivered to those dams with antibodytreatment before delivery (FIG. 10A) developed the disease earlier andmore severely as compared to pups treated with RRV alone (FIG. 10A).

6.3 Antibodies can Induce Cancers

The following tissue sections of pup bile ducts were detected forbinding of anti-NCDV antibody and proliferation (inflammation) cellsusing FITC-conjugated anti-goat IgG antibody, Texas-Red-conjugatedanti-proliferating cell nuclear antigen (PCNA) and immunofluorescentstaining.

1) Saline to mother without RRV infection (Normal);

2) RRV to pups (RRV);

3) Anti-NCDV to mother without RRV infection (Ab);

4) Anti-NCDV to mother and RRV to pups (Ab+RRV).

Goat IgG bound to the proliferating bile duct epithelium cells (FIG. 11:HE stain) were detected on the bile duct of the pups with the treatmentof antibody to mother with and without RRV to pups as mentioned above(FIG. 11: Ab and Ab+RRV of the raw of NCDV). These positive results werecompacted to the immunofluorescent staining of the bile duct of the pupgroups of saline and RRV as negative controls for anti-NCDV-binding(FIG. 11: Saline and RRV of the raw of NCDV).

Further, a marker for proliferation, proliferating cell nuclear antigen(PCNA) was detectable on the proliferating cells of the two groups withanti-NCDV treatment especially the pups with antibody to mother withoutRRV infection, compared to the other two groups (FIG. 11: raw of PCNA).The anti-NCDV antibody also bound to the liver section of a P2 pupstreated with antibody to mother compared to the pups treated withsaline. The raw of overlay showed that the anti-NCDV antibodies bound tothe same cells expressing PCNA.

Aberrant expression of PCNA has been reported in various malignancies.Because the anti-NCDV antibodies stimulated inflammatory cellsexpressing PCNA and many cancers are initiated from inflammation, theantibodies induced during a infection have the potential to inducecancers. This can occur if such antibody stimulation exists persistentlyand eventually leads to an uncontrolled cell growth.

As described in PCT/US2009/039810, the anti-NCDV antibodies bound toN-Acetyl-D-Glucosamine. Therefore, glycan N-Acetyl-D-Glucosamine is apotential biological marker related to inflammation and cancers.

7. Diagnosis Kits for Rotavirus Infection

A sandwich ELISA kit for detection of rotavirus was developed as below.

-   -   a. Capture antibody: the monoclonal anti-VP6 antibody (0.1        mg/ml) (Meridian Life Science, Inc., Saco, Me., USA): 1:200        dilution.    -   b. Detection antibody (primary): the purified goat polyclonal        anti-NCDV antibody (5 mg/ml) (Meridian Life Science, Inc., Saco,        Me., USA): 1:200 dilution.    -   c. Secondary detection antibody: an anti-goat IgG antibody        (Meridian Life Science, Inc., Saco, Me., USA).

Viral loads in the sera from mouse pups infected with RRV were detectedby the ELISA kit and the results are shown in FIG. 12C.

8. Other Drug Candidates for Prevention and Treatment of ViralInfections

8.1. Prevention and Treatment of Rotavirus Viral Infection

Three groups of sucking bulb/c mouse pups were treated at day 1 afterbirth (P1) via oral administration with saline (control group) or drugcandidate followed by challenging at day 2 (P2) with 20 μl (microliter)of rhesus rotavirus (RRV) at the concentration of 1×10⁷ PFU/ml. Thedifferent groups of mice were pre-treated with each 20 μl (microliter)of: 1) saline (RRV group, n=7); 2) the methionine-Zinc (Zn) complex atthe concentration of 2 mg/ml (Met group, n=7); and 3) a formulationconsisted of the Neu5Ac (2 mg/ml) and the methionine-Zinc (Zn) complex(2 mg/ml) (n=6). Mice were kept for 3 weeks after RRV infection. Thecourse of rotavirus infection is described above.

TABLE 7 The results of efficacy test of drug candidates for rotavirus(RRV) infection Not Sick Odds P Drug candidate Sick sick Rate(%) Ratio95% CI value Saline 6 1 86 30 1.47-612  0.03 Metnionine-Zn 1 6 14 0.030.001-0.55  0.03 Neu5Ac + 1 5 17 0.03 0.02-0.68 0.03 Meth-Zn* *Meth-Zn =Metnionine-Zn complex.

Pups pretreated with the drug candidates were not infected compared tocontrol pups pretreated with saline. The results with statistic analysisare concluded in Table 7.

The effective dosages of drug candidates for rotavirus infection forhuman are calculated and listed in Table 8.

TABLE 8 The dosages of drug candidates for rotavirus infection Drugcandidate Low (mg/kg) High (mg/kg) Range (mg/kg) Saline 0.1 20 0.1-20Metnionine-Zn 0.1 20 0.1-20 Neu5Ac + Meth-Zn* 0.1:0.1 20:200.1:0.1-20:20  *Meth-Zn = Metnionine-Zn complex.

8.2 An Animal Model of Later Term Embryo and Newborn Chicks

Four groups of day 16 (E16) chicken embryos were pretreated viaallantois injection with 100 microliter of saline solution containing 1)saline alone (n=6); 2) 20 microliter of 2% diethyl disulfide (n=6); 3)20 microgram of the allium sativum (n=6); and 4) 100 microgram of theNeu5Ac and 100 microgram of the methionine (n=6), followed byinoculation via allantois injection of 100 μl (microliter) of the2009H1N1 influenza virus (California strain, titer: 1:128, diluted 100times with saline) next day. Allantois fluid was collected at 48 hoursafter viral inoculation and viral titers in the fluid were determined bya direct hemagglutination test. The chicken embryos were kept culturingin a 35° C. incubator until the newborn chickens coming out.

Viral titers of 2009H1N1 California strain in allantois fluid (48 hours)are summarized below.

Viral titers of 2009H1N1 (California) virus in allantois fluid (48hours) Chicken embryo 1 2 3 4 5 6 Saline 4+ 4+ 4+ 4+ 4+ − Diethyldisulfide 2+ − − + − − Allium sativum − − − − − − Neu5Ac + + − − − − +Methionine

The group-1 (virus control) newborn chicks looked sick and the newbornchicks of group-2 (diethyl disulfide), group-3 (garlic) and group-4(formula) looked healthy. As summarized in Table 9, diethyl disulfide,allium sativum and the formula significantly inhibited infections oflater term chicken embryos by the 2009 H1N1 and seasonal H1N1 viruses.

TABLE 9 The results of chicken embryo test of drugs against 2009H1N1virus infection Not Sick Odds P Drug candidate Sick sick Rate(%) Ratio95% CI value Saline 5 1 83 ND* ND ND Diethyl disulfide 1 5 17 0.040.002-0.83 0.08 Allium sativum 1 6 14 0.03 0.002-0.68 0.03 Neu5Ac + 1 517 0.04 0.002-0.83 0.08 Methionine *ND = Not Difined.

The animal model of later term embryo (E16-E20) and newborn chicken canbe used but not limited for the screening of anti-influenza virus drugs.

9. Antibodies for Prevention and Treatment of Influenza Infections

Following immune sera were tested for its efficacy on prevention andtreatment of viral infections as described below.

-   -   a. Chicken immune serum against the 2009H1N1 influenza virus        (California strain) (National influenza Center of China CDC).        -   Immunogen: purified 2009H1N1 influenza virus (California            strain).        -   Antibody titer: 1:1280 (10 times dilution to 1:128).    -   b. Goat immune serum against a seasonal H1N1 influenza virus        (Shanghai, 1999) (National influenza Center of China CDC).        -   Immunogen: purified seasonal H1N1 influenza virus (Shanghai,            1999).        -   Antibody titer: 1:128.    -   c. Goat immune serum against a H3N2 influenza virus        (Jiangxi, 2004) (National influenza Center of China CDC).        -   Immunogen: the H3N2 influenza virus (Jiangxi, 2004).        -   Antibody titer: 1:1280 (10 times dilution to 1:128).    -   d. Rabbit immune serum against a H5N1 influenza virus        (Anhui, 2005) (National influenza Center of China CDC).        -   Immunogen: purified H5N1 influenza virus (Anhui, 2005).        -   Antibody titer: 1:1280 (10 times dilution to 1:128).    -   e. Human serum pool containing low level of antibodies to        seasonal-H1N1 influenza virus (China 2009) (National influenza        Center of China CDC).        -   Antibody titer for influenza H1N1, H3N2 and B viruses: 1:5.

All the immune sera except the anti-2009H1N1 (California) sera mentionedabove did not react to the 2009H1N1 (California) influenza virusdetermined by a hemagglutination test.

9.1 A Newborn Mouse Model for the Treatment of Influenza Infection

Three groups of newborn bulb/c pups were inoculated at day 5 (P5) vianasal and oral administration of 30 μl (microliter) of theA/PR/8/34(H1N1) influenza virus (titer: 1:512, diluted 300 times withsaline); and were treated at day 6 (P6) via intraperitoneal injectionwith 100 μl of saline containing 1) saline alone (n=6); 2) 20 μl(microliter) of the chicken anti-2009H1N1 influenza virus serum(antibody titer: 1:128) (n=7); 3) 20 μl (microliter) of the chickenanti-2009H1N1 influenza virus serum pre-treated with 10 μl (microliter,100 μg) of the Neu5Ac (10 mg/ml) (total=20 μl, incubated for 30 minutes)(n=7); and 4) 20 μl (microliter) of the formula consisted of Neu5Ac (200μg) and 2% diethyl disulfide (n=6). Mice were kept for 7 days aftertreatment.

As summarized in Table 10, ⅚ (83%) of the pups treated with saline andall (100%) of the pups treated with the anti-2009H1N1 serum alone weredied at day 2 after viral infection. Only 1/7 (14.3%) of the pupstreated with the formula consisted of anti-2009H1N1 serum+Neu5Ac, and2/7 (28.6) of the pups treated with the formula consisted ofNeu5Ac+methionine were died at day 2 after viral infection. The resultsindicated that the formula consisted of anti-20009H1N1 antibodies andNeu5Ac reduced the death rate of the highly pathogenic A/PR/8/34(H1N1)infection from 83.3% to 14.3% (5.9 folds); and the formula consisted ofNeu5Ac and methionine reduced the death rate of the A/PR/8/34(H1N1)infection from 83.3% to 28.6% (2.8 folds). The data also suggested thathigh dose of the anti-20009H1N1 antibodies alone are harmful rather thanhelpful for the treatment of the A/PR/8/34(H1N1) infection in newbornmouse pups.

TABLE 10 The results of bulb/c mouse test of drugs against theA/PR/8/34(H1N1) virus infection No Death Odds P Drug candidate Deathdeath Rate(%) Ratio 95% CI value Saline 5 1 83.3 ND* ND ND H1N1-Ab.1^(a)7 0 100 ND  ND ND H1N1-Ab.1 + 1 6 14.3 0.03 0.002-0.68 0.03 Neu5AcNeu5Ac + 2 5 28.6 0.08 0.005-1.19 0.10 Methionine *ND = Not Difined.

9.2 A Newborn Mouse Model for the Prevention of Influenza Infection

Eight groups of newborn bulb/c mouse pups were pretreated at day 6 (P6)via intraperitoneal injection with 50 μl of saline solutioncontaining 1) saline alone (control group, n=6); 2) 10 μl (microliter)of the chicken anti-2009H1N1 serum (n=7); 3) 10 μl (microliter) of thegoat anti-seasonal H1N1 serum (n=7); 4) 10 μl (microliter) of the humanserum pool (n=5); 5) 10 μl (microliter) of the goat anti-H3N2 serum(n=5); 6) 10 μl (microliter) of the rabbit anti-H5N1 serum (n=6); 7) 10μl (microliter) of the chicken anti-2009H1N1 influenza virus serumpre-treated with 10 μl (microliter, 300 μg) of the Neu5Ac (30 mg/ml)(incubated for 30 minutes) (n=5); and 8) 10 μl (microliter) of thechicken anti-2009H1N1 influenza virus serum plus 10 μl (microliter) ofthe human serum pool (n=5). The pups were inoculated at day 7 (P7) viaoral administration of 50 μl (microliter) of the A/PR/8/34(H1N1) virus(titer: 1:512, diluted 100 times with saline), and kept for 7 days afterviral infection.

The results are summarized in Table 11. The data indicated that 1) theformula consisted of anti-2009H1N1 antibodies+Neu5Ac or antibodiesagainst H5N1 are significantly effective for the prevention of theA/PR/8/34(H1N1) influenza virus infection; 2) the human serum poolcontaining lower dose of anti-seasonal H1N1 antibodies (H1N1-Ab.3, 1:5)reduced the death rate of the A/PR/8/34(H1N1) infection from 83.3% to40.0% (2.1 folds); 3) the serum containing higher dose of anti-seasonalH1N1 antibodies (H1N1-Ab.2, 1:128) is not effective for the preventionof the A/PR/8/34(H1N1) infection; and 4) the serum containing higherdose of anti-2009H1N1 antibodies (H1N1-Ab.1, 1:128) is not effective orharmful for the prevention of the A/PR/8/34(H1N1) infection of newbornpups.

TABLE 11 The mouse test of antibodies for preventing the A/PR/8/34(H1N1)viral infection No Death Odds Treatment n = death (%) Ratio 95% CI PSaline 6 1 5 (83.3) 1.4  0.07-28.1 1.0  H1N1-Ab.1^(a) 7 0 7 (100)Infinity Infinity Infinity H1N1-Ab.2^(b) 7 0 7 (100) Infinity InfinityInfinity H1N1-Ab.3^(c) 5 3 2 (40.0) 0.13 .008-2.2 0.24 H3N2-Ab 5 0 5(100) Infinity Infinity Infinity H5N1-Ab 6 5 1 (16.7) 0.04 .002-0.830.08 H1N1-Ab.1 + 6 5 1 (16.7) 0.04 .002-0.83 0.08 Neu5Ac H1N1-Ab.1 + 5 23 (60.0) 0.30 0.02-4.91 0.55 Ab.3 ^(a)Chicken anti-2009 H1N1(California, 2009) serum, 1:128; ^(b)Goat anti-seasonal H1N1 (Shanghai,1999) serum, 1:128; ^(c)Human serum pool: anti-H1N1 (China, 2009): 1:5.

The animal models described above can be used but not limited for rapidevaluation of the efficacy and side effect of vaccines and antibodiesamong other ages of animals or people.

9.3 A Newborn Chick Model for Influenza Infection

Five groups of day 16 (E16) chicken embryos were treated via allantoisinjection of each 100 μl (microliter) of 1) saline alone (n=6); 2) thechicken anti-2009H1N1 influenza virus serum (1:128, n=9); 3) the goatanti-seasonal H1N1 serum (1:128, n=8); 4) the human serum pool (1:5,n=6); 5) the chicken anti-2009H1N1 (California) serum (1:128) pretreatedwith 10 μl (microliter, 300 μg) of the Neu5Ac (30 mg/ml) (incubated for30 minutes) (n=8). Next day (E17), chicken embryos were inoculated viaallantois injection of 100 μl (microliter) of the 2009H1N1 influenzavirus (California strain, titer: 1:128, diluted 100 times with saline).The chicken embryos were kept culturing in a 35° C. incubator until thenewborn chicks coming out.

The data indicated that the formula consisted of anti-2009H1N1antibodies+Neu5Ac are significantly effective for the prevention of the2009H1N1 influenza virus infection (Table 12, FIG. 12D); and the othersera containing moderate or high dose of anti-influenza virus antibodies(H1N1-Ab.1 and H1N1-Ab.2) are harmful rather than effective for theprevention of the 2009H1N1 virus infection (FIGS. 12B and 12C) althoughthey have lower death rates (Table 12).

TABLE 12 The results of chicken test of antibodies for preventing2009H1N1 influenza viral infection Not Sick Death Odds Treatment n =sick (%)* (%) Ratio 95% CI P Virus alone** 6 1 5 (83.3) 3 (50.0) 0.830.04-17.0 1.0 H1N1-Ab.1 + Virus 9 1 8 (88.9) 2 (22.2) 1.2 0.06-24.5 1.0H1N1-Ab.2 + Virus 8 1 7 (87.5) 3 (37.5) 1.4 0.07-28.1 1.0 H1N1-Ab.3 +Virus 6 1 5 (83.3) 2 (33.3) 1.0 0.05-20.8 1.0 H1N1-Ab.1 + Neu5Ac 8 7 1(12.5) 1 (12.5) 0.03 .001-0.57 0.03 *including death; **2009H1N1(California) influenza virus; a: chicken anti-2009H1N1(California)serum, 1:128; b: goat anti-seasonal H1N1(Shanghai, 1999) serum, 1:128;c: human serum pool: anti-H1N1 (China, 2009): 1:5.

The animal model of later term embryo (E16-E20) and newborn chicks canbe used but not limited for evaluation of the efficacy and safety ofinfluenza vaccines and antibodies.

10. Experimental Models for Evaluation of Vaccines and Antibodies

10.1 An Animal Model for Rapid Evaluating the Safety of Vaccines andAntibodies

Six groups of day 16 (E16) chicken embryos were treated via allantoisinjection of each 100 μl (microliter) of 1) saline alone (n=10); 2) thechicken anti-2009H1N1 influenza virus serum (n=11); 3) the goatanti-seasonal H1N1 serum (n=11); 4) the human serum pool (n=8); 5) thegoat anti-H3N2 serum (n=10); 6) the rabbit anti-H5N1 serum (n=8); Thechicken embryos were kept culturing in a 35° C. incubator until thenewborn chicks coming out.

The results are summarized in Table 13 and FIG. 13. The chicks treatedwith the anti-2009H1N1 serum (FIG. 14B), the anti-seasonal H1N1 serum(FIG. 13C), and the anti-H5N1 serum (FIG. 13E) looked sick. However, thechicks treated with saline (FIG. 13A), the anti-H3N2 serum (FIG. 13D),and the human serum pool (FIG. 13F) looked healthy. The data indicatedthat moderate or high dose of anti-H1N1 and anti-H5N1 antibodies inducedeither death or severe disease in chicken fetus and newborn chicks.

TABLE 13 The results of chicken test of the efficacy and safty ofantibodies against influenza viruses Not Sick Death Odds Treatment n =sick (%)* (%) Ratio 95% CI P Saline 10 9 1 (10) 1 (10) 1.1 0.06-20.5 1.0H1N1-Ab.1^(a) 11 1 10 (91)  2 (18) 90   4.9-1660 0.0003 H1N1-Ab.2^(b) 112 9 (82) 5 (46) 41 3.09-530  0.002 H1N1-Ab.3^(c) 8 7 1 (13) 1 (13) 1.290.07-24.3 1.00 H3N2-Ab 10 7 3 (30) 3 (30) 3.86 0.33-45.6 0.58 H5N1-Ab 84 4 (50) 1 (13) 9.0 0.81-100  0.14 *including death; ^(a)chickenanti-2009H1N1(California) serum, 1:128; ^(b)goat anti-seasonalH1N1(Shanghai, 1999) serum, 1:128; ^(c)human serum pool: anti-H1N1(China, 2009): 1:5.

10.2 An Animal Model of Guillain-Barre Syndrome

Injection of the anti-2009H1N1 serum, the anti-seasonal H1N1 serum andthe anti-H5N1 serum into E16 chicken embryo as described above inducedthe leg disability of newborn chicks (FIGS. 13B, 13C and 13D) which issimilar to the Guillain-Barre syndrome (GBS) in human. The frequenciesof GBS induced by the anti-influenza virus antibodies are listed inTable 14. The results indicated that the antibodies induced by the2009H1N1 (California) virus is at highest risk for inducing GBS,followed by antibodies induced by the avian H5N1 (Anhui, 2005) virus andthe seasonal-H1N1 (Shanghai, 1999) virus.

TABLE 14 The frequency of Guillain-Barre syndrome induced byanti-influenza antibodies Not GBS Treatment n = sick (%)* OR 95% CI PSaline 10 9 0 (0.0) Infinity Infinity Infinity H1N1-Ab.1^(a) 11 1 8(89)  72  3.84-1350  0.001 H1N1-Ab.2^(b) 11 2 4 (67)  18 1.24-261 0.04H1N1-Ab.3^(c) 8 7 0 (0.0) Infinity Infinity Infinity H3N2-Ab 10 7 0(0.0) Infinity Infinity Infinity H5N1-Ab 8 4 4 (50)    9.0 0.81-100 0.14H1N1-Ab.1 + 6 5 0 (0.0) Infinity Infinity Infinity Neu5Ac*Guillain-Barre syndrome; ^(a)chicken anti-2009H1N1(California) serum,1:128; ^(b)goat anti-seasonal H1N1(Shanghai, 1999) serum, 1:128;^(c)human serum pool: anti-H1N1 (China, 2009): 1:5.

The animal model of later term embryo (E16-E20) and newborn chicks canbe used but not limited for evaluation of the efficacy and safety ofinfluenza vaccines and antibodies, for the pathogenesis study of GBS andfor screening drugs for the prevention and treatment of GBS and sideeffect of vaccines and antibodies.

11. Therapeutic Application of Neu5Ac for Influenza Infection

11.1 Treatment of the Side Effect of Influenza Vaccines and Antibodieswith Neu5Ac

Each 100 μl (microliter) of the sera containing H1N1-Ab.1, -Ab.2 and theH5N1-Ab were incubated with each 10 μl (microliter, 300 μg) of theNeu5Ac (30 mg/ml) for 30 minutes, then were administrated via allantoisinjection into three groups of day 16 (E16) chicken embryos: 1) thechicken anti-2009H1N1 pre-treated serum (n=8); 2) the goat anti-seasonalH1N1 pretreated serum (n=8); and 3) the rabbit anti-H5N1 pre-treatedserum (n=8). The chicken embryos were kept culturing in a 35° C.incubator until the newborn chicks coming out.

The results are summarized in Table 11 and Table 12. The data show thatthe newborn chicks with injection of the immune sera pre-treated withN-acetylneuraminic acid (Neu5Ac) developed neither disorders nor GBS.

11.2 Further, the immune sera pre-treated with Neu5Ac are effective forprevention and treatment of influenza infections caused by the 2009H1N1(California) virus in newborn chicks as described above (Table 10, FIG.12D).

12. In Vivo Proof of a Novel Acting Mechanism of Vaccination andAntibody Therapy

In vitro and in vivo proof of a novel mechanism of vaccination andpassive immunity has been described in PCT/US2007/018258 and U.S.61/278,685.

For further in vivo proof, the blood were collected from the newbornchicks treated with injection of anti-influenza immune sera as mentionedin 10.1 and 11.1 (Table 11); and the chicks were infected at P3 (day 8after serum injection) via nasal drop and oral administration of 100 μl(microliter) of the 2009H1N1 (California) virus (1:32, 2 timesdilution). The chicks were kept for 4 days after viral infection forobservation of influenza symptoms.

TABLE 15 The results of chicken test of the efficacy of antibodiesagainst the 2009 H1N1 influenza virus Not Sick Death Odds Treatment n =sick (%)* (%) Ratio 95% CI P Saline 9 1 8 (89) 1 (11) 64  3.38-12100.003 H1N1-Ab.1^(a) 9 6 3 (33) 3 (33) 0.06 .005-0.76 0.05 H1N1-Ab.2^(b)6 5 1 (17)  0 (0.0) 0.03 0.001-0.50  0.01 H1N1-Ab.3^(c) 7 2 5 (71) 4(57) 0.31 0.02-4.41 0.55 H3N2-Ab 7 1 6 (86) 4 (57) 0.75 0.04-14.6 1.00H5N1-Ab 7 4 3 (43) 3 (43) 0.09 0.007-1.22  0.11 *including death;^(a)chicken anti-2009H1N1(California) serum, 1:128; ^(b)goatanti-seasonal H1N1(Shanghai, 1999) serum, 1:128; ^(c)human serum pool:anti-H1N1 (China, 2009): 1:5.

As summarized in Table 15, at day 3 after virus infection, the newbornchicks with injection of the sera containing 2009H1N1-Ab ( 6/9, 63%),seasonal H1N1-Ab (⅚, 83%) and the H5N1-Ab ( 4/7, 57%) eight days agowere not infected by the 2009H1N1 (swine) virus. However, the newbornchicks with injection of the 2009H1N1 immune serum ( 3/9, 33%), the H3N2immune serum ( 4/7, 57%) or the H5N1 immune serum ( 3/7, 43%) were diedat day 3 after virus infection suggesting that those antibodies couldcause worse viral infection in chick infants.

13. Identification of Glycan-Related Biological Markers

Binding of WGA to human healthy and disease tissues was detected with atissue array chip consisted of FDA normal human organs (ArrayI+II)+bonus malignat samples (Immgenex, San Diego, USA). The resultsshowed that majority of normal human organs except bone marrow, salivarygland and hypophysis do not or weakly express N-Acetyl-D-Glucosamine,while following cancer tissues strongly express N-Acetyl-D-Glucosamine:malignant melanoma, brain malignant oligodendroglioma, kidney clear cellcarcinoma, skin basal cell carcinoma of head, throat carcinoma,Hodgkin's lymphoma of supraclavicular, colon intermediate gradeinterstitialoma, thyoid medullary carcinoma and skin squamous cellcarcinoma of left chest wall.

14. Prevention and Treatment of Influenza Infection of A549 Cells

Following drug candidates were tested.

1) the Neu5Ac at the concentration of 3 mg/ml;

2) the Neu5Ac methyl ester (Neu5AcMe) at the concentration of 3 mg/ml;

3) the methionine at the concentration of 3 mg/ml;

4) formula-1 consisted of the Neu5Ac (2 mg/ml) and Diethyl disulfide(2%);

5) formula-2 consisted of the Neu5AcMe (2 mg/ml) and the Diethyldisulfide (2%);

50 μl (microliter) of each drug candidate was added into monolayer ofthe human lung adenocarcinoma epithelial cells (A549 cell line) in a12-well plate, incubated for one hour, discarded the supernatant; thenthe cells were challenged with 100 μl (microliter) of 2009 influenza AH1N1 virus (California strain, 1:128, 50 times dilution), the seasonalH1N1 virus (Brisbane/59/2007, 1:256, 100 times dilution) or H3N2 virus(Brisbane/10/2007, 1:128, 50 times dilution) for one hour, discarded thesupernatant and added culture medium containing 100 μl of each drugcandidate as mentioned above. Two wells with medium alone withoutaddition of drugs were used as controls. The culture supernatant wascollected at 24 and 48 hours and used for a hemagglutination inhibitiontest. The experiment was performed duplicate.

The positive result (inhibiting effect) of the hemagglutinationinhibition test was observed as RBC pellet at the bottom of a well;negative result (no inhibition) was observed as no RBC or partial pelletat the bottom of a well. The results are summarized below.

a. Infection of A549 Cells with 2009 H1N1 Influenza A Virus (CaliforniaStrain).

Well 1-2 3-4 5-6 7-8 9-10 11-12 Hours Treatment Medium Neu5Ac Neu5AcMeMet* Frm-1** Frm-2 Viral 2+ − + − − − 24 titer 4+ + 2+ + − + 48 *Met =methionine; and **Frm = formula as mentioned above.

b. Infection of A549 Cells with Seasonal H1N1 (Brisbane) Influenza AVirus.

Well 1-2 3-4 5-6 7-8 9-10 11-12 Hours Treatment Medium Neu5Ac Neu5AcMeMet* Frm-1** Frm-2 Viral 4+ − − − − + 24 titer 4+ + + 2+ + 2+ 48

c. Infection of A549 Cells with Influenza A Virus H3N2 (Brisbane)Strain.

Well 1-2 3-4 5-6 7-8 9-10 11-12 Hours Treatment Medium Neu5Ac Neu5AcMeMet* Frm-1** Frm-2 Viral 3+ + + − + + 24 titer 4+ 2+ 2+ + 2+ 3+ 48 *Met= methionine; and **Frm = formula as mentioned above.

The above results show that in the A549 cellular culture system, thedrug candidates inhibited the infection of influenza virus 2009 H1N1,seasonal H1N1 and H3N2.

15. Prevention and Treatment of Influenza Infection of Mice

15.1 Antibodies from Pregnant Mother Induced Disease in Fetus orNewborns

Human immune sera collected from subjects either with influenzainfection or immunized with influenza vaccines were injected intopregnant bulb/c mouse dams via intraperitoneal injection at E19 (Table8). The titers of those sera were adjusted to 1:128 and 200 microliterof each serum was used. Human serum pool consisted sera from 5 healthyindividuals and the serum from a RSV infected subject were used ascontrols. The titer of the human serum pool to seasonal influenza H1N1,H3N2 and B viruses was 1:5. The death rates of the fetus or newbornsdelivered to those dams are summarized in Table 16.

TABLE 16 The death rates of fetus or newborns Immune Serum Death Odds POne injection at E19 N = Deaths Rate(%) Ratio 95% CI value Hum* serumpre-vaccinization 12 0 0.00 Infinity Infinity Infinity Human anti-RSV(infection) 15 1 6.70 0.93  0.05-16.4 1.00 Hum anti-09H1N1 post-vaccine12 6 50.0 14.0 1.37-143 0.02 Hum anti-S-H1N1** post-vaccine 14 3 21.43.82  0.35-42.0 0.33 Hum anti-H5N1 post-infection 18 9 50.0 14.01.51-130 0.009 Rabbit anti-H5N1 12 6 50.0 14.0 1.37-143 0.02 *Hum =human **S = seasonal.

The results and the animal model provide direct evidence that a highlevel of anti-2009H1N1 and anti-H5N1 antibodies can cause severe sideeffect even death in fetuses and newborns. Furthermore, this findingsuggests that vaccinating pregnant mothers with the 2009H1N1 (swine)influenza vaccine or a H5N1 vaccine is risky for the fetuses andnewborns.

15.2 Treatment of Influenza Infection of Newborn Mice

Four groups of newborn bulb/c pups were inoculated at day 5 (P5) vianasal and oral administration of 20 μl (microliter) of theA/PR/8/34(H1N1) influenza virus strain (titer: 1:256, diluted 200 timeswith saline); and were treated at day 2, 3, 4 and 5 via intraperitonealinjection with 100 μl of saline containing 1) saline alone (n=10); 2)150 microgram of Neu5AcMe; 3) 30 μl of 2% diethyl disulfide plus 200 μgof the Neu5AcMe (n=10); and 4) 30 μl of 2% diethyl disulfide plus 200 μgof the Neu5Ac (n=10). Mice were kept for 10 days after treatment.

9/10 (90%) of mice treated with saline alone died at day 3 or day 4while 8/10 (80%) of the pups treated with diethyl disulfide+Neu5AcMe and5/10 (50%) of the pups treated with either Neu5AcMe or diethyldisulfide+Neu5Ac survived. The data indicated that the formulasconsisted of diethyl disulfide+Neu5AcMe (OR=0.03, 95% CI=0.002-0.37,p=0.005) are significantly effective for the treatment of a severeA/PR/8/34(H1N1) influenza infection.

Other embodiments besides the above may be articulated as well. Theterms and expressions therefore serve only to describe the disclosure byexample only and not to limit the disclosure. It is expected that otherswill perceive differences, which while differing from the foregoing, donot depart from the spirit and scope of the disclosure herein describedand claimed. All patents, patent publications, and other referencescited herein are incorporated herein by reference in their entirety.

What is claimed is:
 1. A method of treating or preventing viral diarrheacaused by rotavirus, the method comprising administering to a patientexposed to or suspected of exposure to rotavirus, an effective dose ofthe composition comprising: N-acetylneuraminic acid, and Methionine;wherein the effective dose of the composition comprises 0.1 mg to about20 mg of N-acetylneuraminic acid per kg body weight of the patient and0.1 mg to about 20 mg of methionine per kg body weight of the patient,and wherein the ratio of the N-acetylneuraminic acid to the methioninein the effective dose of the composition is 1:1.
 2. The method of claim1, wherein the administering comprises oral or injective administration.3. The method of claim 1, wherein the methionine is complexed with zinc.